Preparation of antibody or an antibody fragment targeted immunoliposomes for systemic administration of therapeutic or diagnostic agents and uses thereof

ABSTRACT

A method of preparing an antibody- or antibody fragment-targeted cationic immunoliposome or polymer complex comprises the steps of (a) preparing an antibody or antibody fragment; (b) mixing said antibody or antibody fragment with a cationic liposome to form a cationic immunoliposome or with a cationic polymer to form a polyplex; and (c) mixing said cationic immunoliposome or said polyplex with a therapeutic or diagnostic agent to form said antibody- or antibody fragment-targeted cationic immunoliposome or polymer complex.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/798,296, filed May 11, 2007, now U.S. Pat. No. 9,034,330, issued May19, 2015, which is a continuation-in-part of U.S. application Ser. No.10/113,927, filed Apr. 2, 2002, now U.S. Pat. No. 7,780,882, which is acontinuation-in-part of U.S. application Ser. No. 09/914,046, filed Oct.1, 2001, now U.S. Pat. No. 7,479,276. U.S. application Ser. No.09/914,046, is a U.S. National Phase Application under 35 U.S.C. §371 ofPCT/US00/04392, filed Feb. 20, 2000, which claims the benefit of U.S.Provisional Application No. 60/121,133, filed Feb. 22, 1999. U.S.application Ser. No. 10/113,927 also claims the benefit of U.S.Provisional Application No. 60/280,134, filed Apr. 2, 2001. U.S.application Ser. No. 11/798,296 also claims the benefit of U.S.Provisional Application No. 60/800,163, filed May 15, 2006 and60/844,352, filed Sep. 14, 2006. The disclosures of each of theseapplications are incorporated by reference herein in their entiretiesfor all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention provides a method of making antibody- or antibodyfragment-targeted immunoliposomes and antibody- or antibodyfragment-targeted polymers useful for the systemic delivery of moleculesto treat diseases. The liposome and polymer complexes are useful forcarrying out delivery of small molecules, as well as targeted genedelivery and efficient gene expression after systemic administration.The specificity of the delivery system is derived from the targetingantibodies or antibody fragments.

2. Related Art

The ideal therapeutic for cancer would be one that selectively targets acellular pathway responsible for the tumor phenotype and would benontoxic to normal cells. To date, the ideal therapeutic remains justthat—an ideal. While cancer treatments involving gene therapy havesubstantial promise, there are many issues that need to be addressedbefore this promise can be realized. Perhaps foremost among the issuesassociated with macromolecular treatments is the efficient delivery ofthe therapeutic molecules to the site(s) in the body where they areneeded. The ideal delivery vehicle would be one that could besystemically administered and then home to tumor cells wherever theyoccur in the body. A variety of delivery systems (“vectors”) have beentried, including viruses and liposomes. The infectivity that makesviruses attractive as delivery vectors also poses their greatestdrawback. Residual viral elements can be immunogenic, cytopathic orrecombinogenic. The generation of novel viruses with new targets forinfection also raises the theoretical possibility that, once introducedinto patients, these viruses could be transformed via genetic alterationinto new human pathogens. Consequently, a significant amount ofattention has been directed at non-viral vectors for the delivery ofmolecular therapeutics. The liposome approach offers a number ofadvantages over viral methodologies for gene delivery. Mostsignificantly, they lack immunogenicity. Moreover, since liposomes arenot infectious agents capable of self-replication, they pose no risk ofevolving into new classes of infectious human pathogens.

Targeting cancer cells via liposomes can be achieved by modifying theliposomes so that they selectively deliver their payload to tumor cells.Surface molecules can be used to target liposomes to tumor cells,because the molecules that decorate the exterior of tumor cells differfrom those on normal cells. For example, if a liposome has the proteintransferrin (Tf) or an antibody that recognizes transferrin receptor(TfR) on its surface, it will home to cancer cells that have higherlevels of the TfR. Such liposomes designed to home to tumors have beenlikened to “smart” bombs capable of seeking out their target.

Failure to respond to therapy represents an unmet medical need in thetreatment of many types of cancer, including prostate cancer. Often whencancer recurs, the tumors have acquired increased resistance toradiation or chemotherapeutic agents. The incorporation into currentlyused cancer therapies of a new component which results inradio-/chemo-sensitization would have immense clinical relevance. Oneway in which such sensitization could be achieved is via gene therapy(i.e., delivery of a gene the expression of which results in increasedsensitization). In PCT patent application WO 00/50008 (published 31 Aug.2000), incorporated herein by reference, we provided proof-of-principlethat an anti-transferrin receptor single chain antibody (TfRscFv) can bechemically conjugated to a cationic liposome. Moreover, this TfRscFvdirected liposome delivery system can deliver genes and other moleculessystemically and specifically to tumors.

Immunoliposomes and Cationic Polymers as Gene Transfer Vehicles

As noted above, some of the problems associated with using viral vectorscould be circumvented by non-viral gene transfer vectors. Progress hasbeen made toward developing non-viral, pharmaceutical formulations ofgenes for in vivo human therapy, particularly cationic liposome-mediatedgene transfer systems (31, 32). Cationic liposomes are composed ofpositively charged lipid bilayers and can be complexed to negativelycharged, naked DNA by simple mixing of lipids and DNA such that theresulting complex has a net positive charge. The complex can be boundand taken up by cells in culture with moderately good transfectionefficiency (33). Features of cationic liposomes that make them versatileand attractive for DNA delivery include: simplicity of preparation; theability to complex large amounts of DNA; versatility in use with anytype and size of DNA or RNA; the ability to transfect many differenttypes of cells, including non-dividing cells; and lack of immunogenicityor biohazardous activity (reviewed in 34, 35). More importantly from theperspective of human cancer therapy, cationic liposomes have been provento be safe and efficient for in vivo gene delivery (33, 34, 36). Atleast 99 clinical trials have been approved using cationic liposomes forgene delivery (37), and liposomes for delivery of small moleculetherapeutics (e.g., antifungal agents) are already on the market.

Researchers also have considered the suitability of cationic polymers astransfer vectors for delivery of therapeutic agents in vivo. Forexample, Polyethyleneimine (PEI) is the organic macromolecule with thehighest cationic-charge-density potential, and a versatile vector forgene and oligonucleotide transfer in vitro and in vivo, as firstreported by Boussif et al. (66). Since then, there has been a flurry ofresearch aimed at this polycation and its role in gene therapy (73).Cell-binding ligands can be introduced to the polycation to 1) targetspecific cell types and 2) enhance intracellular uptake after bindingthe target cell (13). Erbacher et al. (67) conjugated theintegrin-binding peptide 9-mer RGD via a disulfide bridge and showedphysical properties of interest for systemic gene delivery.

The transfection efficiency of both cationic liposomes and cationicpolymers, such as PEI, can be increased dramatically when they bear aligand recognized by a cell surface receptor. Receptor-mediatedendocytosis represents a highly efficient internalization pathwaypresent in eukaryotic cells (38, 39). The presence of a ligand on aliposome facilitates the entry of DNA into cells through initial bindingof ligand by its receptor on the cell surface followed byinternalization of the bound complex. Transferrin receptor (TfR) levelsare elevated in various types of cancer cells including, but not limitedto, breast, pancreatic, head and neck, and prostate cancers (40), eventhose prostate cell lines derived from human lymph node and bonemetastases (40-43). Elevated TfR levels also correlate with theaggressive or proliferative ability of tumor cells (44). Therefore, TfRis a potential target for drug delivery in the therapy of malignant cellgrowth (45, 46). In our laboratory, we have preparedtransferrin-complexed cationic liposomes with tumor cell transfectionefficiencies in SCCHN of 60%-70%, as compared to only 5-20% by cationicliposomes without ligand (47). Also see published PCT patent applicationWO 00/50008.

In addition to the use of ligands that are recognized by receptors ontumor cells, specific antibodies also can be attached to the liposomesurface (48) enabling them to be directed to specific tumor surfaceantigens (including but not limited to receptors) (49). These“immunoliposomes,” especially the sterically stabilized immunoliposomes,can deliver therapeutic drugs to a specific target cell population (50).Parks et al. (51) found that anti-HER-2 monoclonal antibody (MAb) Fabfragments conjugated to liposomes could bind specifically to a breastcancer cell line, SK-BR-3, that overexpresses HER-2. The immunoliposomeswere found to be internalized efficiently by receptor-mediatedendocytosis via the coated pit pathway and also possibly by membranefusion. Moreover, the anchoring of anti-HER-2 Fab fragments enhancedtheir inhibitory effects. More recently, Park et al. (23) used ananti-HER-2 immunoliposome composed of long circulating liposomeschemically conjugated to anti-HER-2 monoclonal antibody scFv fragmentsto deliver doxorubicin to breast cancer tumors even though HER-2 was notover-expressed. A number of other studies have been published which haveemployed antibodies against tumor specific antigens coupled toliposomes, primarily sterically stabilized liposomes, to target tumorcells for delivery of prodrugs and drugs in vitro or in vivo (52-56).These studies demonstrated the utility of immunoliposomes fortumor-targeting drug delivery. The combination of cationic liposome-genetransfer and immunoliposome techniques appears to be a promising systemfor targeted gene therapy and is the subject of this application.

Progress in biotechnology has allowed the derivation of specificrecognition domains from MAb (57). The recombination of the variableregions of heavy and light chains and their integration into a singlepolypeptide provides the possibility of employing single-chain antibodyderivatives (designated scFv) for targeting purposes. Thus, a scFv basedon the anti-TfR MAb 5E9 (52) contains the complete antibody binding sitefor the epitope of the TfR recognized by this MAb as a singlepolypeptide chain of approximate molecular weight 26,000. This TfRscFvis formed by connecting the component VH and VL variable domains fromthe heavy and light chains, respectively, with an appropriately designedpeptide. The peptide bridges the C-terminus of the first variable regionand N-terminus of the second, ordered as either VH-peptide-VL orVL-peptide-VH. The binding site of an scFv can replicate both theaffinity and specificity of its parent antibody combining site.

The TfRscFv has advantages in human use over the Tf molecule itself oreven an entire MAb to target liposomes or cationic polymers to cancercells with elevated levels of the TfR for a number of reasons. First,the size of the scFv (˜28 kDa) is much smaller than that of the Tfmolecule (˜80 kDa) or the parental MAb (˜150 kDa). ThescFv-liposome-therapeutic agent complex or scFv-polymer-therapeuticagent complex thus may exhibit better penetration into small capillariescharacteristic of solid tumors. Second, the smaller scFv also haspractical advantages related to its production as a recombinant protein.Large scale production of the TfRscFv will be required for the therapyenvisioned in this invention to be taken into eventual human trials.Third, the scFv is a recombinant molecule (not a blood product like TOand, therefore, presents no issues related to potential contaminationwith blood borne pathogens. Additional advantages of using the TfRscFvrelate to the fact that Tf interacts with the TfR with high affinityonly after the ligand is loaded with iron. Large-scale production ofliposomes containing iron-loaded Tf may present practical challenges.Thus, use of TtRscFv enables the tumor cell TfR to be targeted by aliposomal therapeutic complex that does not contain iron (itselfimplicated in cancer (58)). Fourth, without the Fc region of the MAb,the problem of non-antigen-specific binding through Fc receptors iseliminated (57).

p53 Tumor Suppressor Gene and the Pathogenesis of Prostate Cancer

The tumor suppressor gene p53 plays a crucial role in diverse cellularpathways including those activated in response to DNA damage, such asDNA repair, regulation of the cell cycle and programmed cell death(apoptosis) (1). Malfunctions of these critical cell pathways areassociated with the process of tumorigenesis. Loss of functional p53,which has been implicated in over 60% of human cancers, can occur eitherthrough mutations in the p53 gene itself (the most common occurrence),or through other mechanisms such as amplification of the MDM-2 gene(found in certain sarcomas, and other cancers), or association of p53with the E6 protein of human papilloma virus (which likely plays a rolein cervical carcinoma) (2).

The loss of p53 function is of relevance to a broad array of cancertypes, with non-functional p53 associated with, for example, 15-50% ofbreast cancer, 25-70% of metastatic prostate cancer, 25-75% of lungcancer, and 33-100% of head and neck cancers (3). The presence of mutantp53 also has been associated with an unfavorable prognosis for manyhuman cancers including lung, colon, and breast (3), and mutant p53 israrely found in some of the most curable forms of cancer e.g., Wilm'stumor, retinoblastoma, testicular cancer, neuroblastoma and acutelymphoblastic leukemia (4). In addition, p53 protein transcriptionallyregulates genes involved in angiogenesis, a process required for solidtumor growth (5). Volpert et al. have proposed that development of theangiogenic phenotype for these tumors requires the loss of both p53alleles (6).

Since it appears that most anti-cancer agents work by inducing apoptosis(20), inhibition of or changes in this pathway may lead to failure oftherapeutic regimens. A direct link has been suggested between mutationsin p53 and resistance to cytotoxic cancer treatments (both chemo- andradiotherapy (21)). It has also been suggested that the loss of p53function may contribute to the cross-resistance to anti-cancer agentsobserved in some tumor cells (22).

Restoration of p53 function could, therefore result in sensitization ofprimary prostate tumors and even metastases to radio-/chemo-therapy. Theintroduction of wtp53 has been reported to suppress, both in vitro andin mouse xenograft models, the growth of various types of malignancies,e.g., prostate (23,24), head and neck (25,26), colon (27), cervical (28)and lung (15,29) tumor cells. However, p53 alone, while being able topartially inhibit tumor growth, has not been shown to be able toeliminate established tumors. Significantly, however, we havedemonstrated that the combination of systemically delivered liposome-p53and radiation led to complete long-term tumor regression of establishedhead and neck xenograft tumors (25,30).

In summary, the implication of the p53 gene in a significant fraction ofhuman cancers makes it one of the premiere candidates for cancer genetherapy. Based on a growing body of evidence related to p53 functions,effective restoration of these functions in tumor cells might beexpected to re-establish normal cell growth control, restore appropriateresponses to DNA-damaging agents (e.g., chemotherapy and radiotherapy),and to impede angiogenesis.

The sensitization of tumors to chemotherapy and radiation could lowerthe effective dose of both types of anticancer modalities,correspondingly lessening the severe side effects often associated withthese treatments. Until now the vast majority of p53 gene therapyprotocols have employed wtp53 gene replacement alone. Based upon thecurrent literature and our data (30, 59), it appears that wtp53replacement alone, while able to inhibit tumor growth to some extent, isinsufficient to eliminate tumors long term. Therefore, it appears that acombinatorial approach involving both standard therapy and targeted genetherapy has substantial promise as a novel and more effective clinicalmodality for cancer treatment. Moreover, the demonstrated tumor cellselectivity of our systemically delivered ligand-liposome wtp53 complexindicates the potential of this method to sensitize even the distantmicrometastases that are the ultimate cause of so many prostate cancerdeaths.

Components of intracellular signaling pathways including, but notlimited to receptor tyrosine kinases (RTKs) and non-receptor tyrosinekinases (non-RTKs), are crucial mediators of many critical pathwaysincluding cell proliferation, differentiation, migration, angiogenesis,cell cycle regulation etc. (Baselga, Science 312, 1175-1178 (2006),Arora and Scholar, J Pharmacol Exp Ther 315, 971-979 (2005), Krause andVan Etten N Eng J Med 353, 172-187 (2005)). Many of these crucialpathways are deregulated in cancer cells. Thus, RTKs and non-RTKs aregood targets for cancer therapeutics. One class of such therapeutics aresmall molecules including, but not limited to those that target growthfactor receptors and thus affect these signaling pathways (Imai andTakoka, Nature Reviews: Cancer 6, 714-727 (2006)). These inhibitorscompete with ATP (ATP mimetics) and inhibit kinase activity. One of thefirst successful small molecule inhibitors is Imatinib mesylate(GLEEVEC®). This small molecule inactivates the kinase activity ofBCR-ABL fusion protein in CML (Druker Trends in Molecular Medicine 8,S14-S18 (2002)), and has shown significant efficacy in the treatment ofpatients with Philadelphia chromosome positive CML. It is also aninhibitor of other TKs, including KIT and PDGFRα and PDGFRβ KIT isinvolved in metastatic GISTs and the two platelet derived growth factorreceptors are involved in tumors such as glioblastoma anddermatofibrosarcoma protuberans. Because it is a member of the EGFsuperfamily, EGFR is also a logical target for small moleculeinhibitors. Gefitinib (IRESSA®) (Herbst et al Nature reviews Cancer 4,9560965 (2004)) and erlotinib (TARCEVA®) (Minna and Dowell NatureReviews Drug Discovery Suppl. S14-S15 (2005)) selectively inhibit EGFRand have shown efficacy against EGFR expressing cancers such as NSCLCand squamous cell carcinomas of the head and neck. They have also shownefficacy in Phase II trials in combination with chemotherapeutic agents.The combination of erlotinib and chemotherapeutic agent gemcitibine (ananti-metabolite) has been approved for use in treating advancedpancreatic cancer. Several Phase III trials of Gefitinib are on going(Chai and Grandis Current Treat Opin Oncol 7, 3-11 (2006)).

Small Molecule agents can translocate through the plasma membrane andinteract with the cytoplasmic domain of the cell surface receptors andintracellular signaling molecules. Thus, small molecules are also beingdeveloped that affect cancer cell proliferation and survival byinhibiting RAS prenylation, RAF-MEK kinase, PI3Kinase, the mTOR pathway(the mammalian target of rapamycin, and even heat shock protein 9). Theycan also affect cell adhesion and invasion by inhibiting SRC kinase ormatrix metalloproteinases. Inhibition of vascular endothelial growthfactor (VEGF) by small molecules can also inhibit neovascularization oftumors.

A new type of small molecule agent, Sorafenib (Nexavar) exerts itsinhibitory effect on different isoforms of RAF serine kinase as well asvarious RTKs (VEGF, EGFR, and PDGF) (Arora and Scholar, J Pharmacol ExpTher 315, 971-979 (2005)). This “dual-action” kinase inhibitor showsbroad-spectrum anti-tumor activity by inhibiting tumor proliferation andangiogenesis (Marx Science 308, 1248-1249 (2005)). Sunitinib malate(SUTENT®) is also a multitargeted TK inhibitor of VEGF, PDGFR, KIT andFLIT3 (Marx Science 308, 1248-1249 (2005)). Potential targets for smallmolecule agents have also been identified in the ubiquitin-proteosomepathway which is crucial in cell cycle arrest and apoptosis (programmedcell death). A selective, reversible inhibitor of the chymotrypticprotease in the 26S proteosome, Bortezomb (Velcade), has been reportedto be effective against various cancers, particularly hematologicalmalignancies.

The anti-EGFR TK inhibitors are synthetic chemicals of ˜500 Da that areadministered orally, with half-lives of ˜46 hours for (IRESSA®) and ˜36hours for TARCEVA®. Because they are administered orally rather thanintravenously, plasma concentrations at the same dose of the smallmolecule therapeutic can vary between patients (Dancy and SausvilleNature Rev Drug Discov 2, 116-124 (2003)). This is a disadvantage ofthese agents as currently used. Thus encapsulating small molecule agentsin a tumor-targeting delivery complex that can be administeredintravenously consistently at the same dose, such as that of thisinvention, would improve their use as therapeutic agents. Furthermore,encapsulation in such a ligand-liposome complex would protect the smallmolecule agents from degradation further enhancing their efficacy.Untargeted orally administered small molecule agents are not specificfor tumor cells, a fact which increases the risk of normal cell toxicityand adverse side effects. While these side effects are generally mild(e.g. rash, acne, dry skin and pruritis) the gastrointestinal toxities(nausea, vomiting, anorexia and particularly diarrhea) can be doselimiting. The most common side effect, skin rash, is possibly due tonon-specific effects on the target kinase in the epidermis (Herbst et alClin Lung Caner 4, 366-369 (2003)). Thus, delivery by a tumor cellspecific agent could decrease this problem. The most severe toxicityreported to date is with IRESSA® (gefitinib): interstitial pneumonitis,a form of pneumonia characterized by non-infectious inflammation andfibrosis in the lower respiratory tract. Over 170 patients have died ofthis disease after treatment with IRESSA® (Arora and Scholar, JPharmacol Exp Ther 315, 971-979 (2005)). Recently, chest CT andradiographic imaging has shown that gefitinib-related interstitial lungdisease is similar to that of pulmonary damage caused by conventionalantineoplastic agents and there may be a direct cytotoxic effect.Therefore, the use of a tumor cell specific agent to deliver IRESSA®, orany small molecule agent, directly to the tumor cells might provide asolution to the problem of interstitial pneumonitis and other sideeffects. Moreover, direct delivery to the site where needed (primary andmetastatic disease), including in the brain, by the method of thisinvention would also result in a decrease in the dose required foreffective treatment, a further benefit currently not possible.

BRIEF SUMMARY OF THE INVENTION

In accordance with this invention a variety of immunoliposomes andpolymer complexes have been constructed that are capable oftumor-targeted, systemic delivery of a variety of types of therapeuticmolecules for use in treating human diseases. The antibody- or antibodyfragment-targeted immunoliposomes or polymer complexes are made via asimple and efficient non-chemical conjugation method. These complexesare equally as effective as, or more effective than, similar complexesprepared by chemical conjugation of the antibody or antibody fragment tothe liposome or polymer complex. If an antibody fragment is used, theresultant complex is capable of producing a much higher level oftransfection efficiency than the same liposome-therapeutic agent orpolymer-therapeutic agent complex bearing the complete antibodymolecule.

In accordance with the present invention, the single chain protein isnot chemically conjugated to the liposome or polymer. Rather, theantibody- or scFv-liposome-therapeutic or diagnostic agent complex orthe antibody- or scFv-polymer-therapeutic or diagnostic agent complex isformed by simple mixing of the components in a defined ratio and order.The antibody- or antibody fragment is complexed (e.g., associated, forexample via a charge-charge interaction) directly with the liposome. Inone embodiment, the antibody or single chain protein first is mixed withthe cationic liposome or the polymer at a protein:lipid ratio in therange of about 1:20 to about 1:40 (w:w) or protein:polymer ratio in therange of about 0.1:1 to 10:1 (molar ratio). The antibody- or antibodyfragment-liposome or antibody- or antibody fragment-polymer then ismixed with a desired therapeutic or diagnostic agent, such as nucleicacid, at a ratio in the range of about 1:10 to 1:20 (μg therapeutic ordiagnostic agent:nmole total lipid) or about 1:1 to 1:40 (ug therapeuticor diagnostic agent:nmole polymer) and incubated for 10-15 minutes atroom temperature. In embodiments where the therapeutic or diagnosticagent is a small molecule, the antibody- or antibody fragment-liposomeor antibody- or antibody fragment-polymer is mixed with the smallmolecule at a molar ratio in the range of about 0.2:7 to about 14:7(small molecule:liposome/polymer complex), suitably at a molar ratio ofabout 2.8:7 or about 7:7 (small molecule:liposome/polymer complex).

The resultant therapeutic or diagnostic agent-antibody-liposome ortherapeutic agent-antibody-polymer complex can be administered to amammal, preferably a human, to deliver the agent to target cells in themammal's body. Desirably the complexes are targeted to a site ofinterest, which can be a cell, including a cancer cell or a non-cancercell. The targeting agent is an antibody or antibody fragment, which inone exemplary embodiment binds to a transferrin receptor, and the targetcell is a cell which expresses or contains the target site of interest.If the antibody or antibody fragment binds to a transferrin receptor,the target cell is a cell which expresses a transferrin receptor. Thetherapeutic agent can be a small molecule, a nucleic acid, including aDNA molecule and suitably a DNA molecule which encodes a wild type p53molecule, Rb molecule or Apoptin molecule or an antisense HER-2. Thecomplexes, for example in a therapeutic composition, can be administeredsystemically, preferably intravenously.

In an additional embodiment, the present invention provides methods ofpreparing an antibody- or antibody fragment-targeted cationicimmunoliposome complex comprising preparing an antibody or antibodyfragment; mixing the antibody or antibody fragment with a cationicliposome to form a cationic immunoliposome, wherein the antibody orantibody fragment is not chemically conjugated to the cationic liposome(but is directly complexed/associated with the liposome); and mixing thecationic immunoliposome with a small molecule to form said antibody- orantibody fragment-targeted-cationic immunoliposome complex. In suitableembodiments, the antibody fragment is a single chain Fv fragment, suchas an anti-transferrin receptor single chain Fv (TfRscFv).

Suitable lipids useful in preparing the small molecule-comprisingcationic immunoliposome complexes of the present invention includemixtures of one or more cationic lipids and one or more neutral orhelper lipids. Suitably, the antibody or antibody fragment is mixed withsaid cationic liposome at a ratio in the range of about 1:20 to about1:40 (w:w). In embodiments, the cationic liposomes comprise a mixture ofdioleoyltrimethylammonium phosphate withdioleoylphosphatidylethanolamine and/or cholesterol; or a mixture ofdimethyldioctadecylammonium bromide withdioleoylphosphatidylethanolamine and/or cholesterol. In furtherembodiments, the cationic immunoliposome is mixed with the smallmolecule at a molar ratio in the range of about 0.2:7 to about 14:7(small molecule:immunoliposome), suitably at a molar ratio of about 1:7to about 12:7, about 1:7 to about 10:7, about 2:7 to about 9:7, about4:7 to about 8:7, about 5:7 to about 8:7 or about 7:7 (smallmolecule:immunoliposome).

Small molecules for use in the practice of the present inventionsuitably will have a molecular weight of less than about 5000 Daltons,more suitably less than about 1000 Daltons, for example about 300 toabout 700 Daltons. In additional embodiments, the small molecules haveat least one pKa in the range of about 2 to about 9. Suitable smallmolecules for use in the practice of the present invention includeanticancer small molecules, including, but not limited to, GMC-5-193,YK-3-250, imatinib mesylate, erlotinib hydrochloride, sunitinib malate,gefitinib and analogs and derivatives thereof. In a further embodiment,the present invention provides small molecule-comprising cationicimmunoliposome complexes prepared by the methods described herein.

In an additional embodiment, the present invention provides antibody- orantibody fragment-targeted cationic immunoliposome complexes comprisinga cationic liposome, an antibody or antibody fragment, and a smallmolecule, wherein the antibody or antibody fragment is not chemicallyconjugated to said cationic liposome (but is directlyassociated/complexed with the liposome). The small molecule may beencapsulated within the cationic liposome, contained within ahydrocarbon chain region of the cationic liposome, associated with aninner or outer monolayer of the cationic liposome (including thehead-group region), or any combination thereof.

The present invention also provides methods of treating a patientsuffering from, or predisposed to, a disease state, such as, but notlimited to, cancer, comprising administering the smallmolecule-comprising cationic immunoliposome complexes of the presentinvention to the patient. Suitably the complexes are administered viaintravenous administration. Alternatively, the complexes can bedelivered via other routes of administration, such as intratumoral,intralesional, aerosal, percutaneous, endoscopic, topical, oral, orsubcutaneous administration. In embodiments where the patient issuffering from or predisposed to cancer, the methods of the presentinvention can further comprise administering radiation or achemotherapeutic agent to the patient, either before, during, or after(e.g., at least 12 hours before, at least 12 hours after, or at the sametime) administration of the cationic immunoliposome complex. Suitablechemotherapeutic agents include, but are not limited to, doxorubicin,cisplatin, mitoxantrone, (docetaxel) TAXOTERE® and CDDP. The presentinvention also provides methods of enhancing the effectiveness of achemotherapeutic agent comprising administering the smallmolecule-comprising cationic immunoliposomes of the present invention inconjunction with the chemotherapeutic agent to a patient, either before,during or after (e.g., at least 12 hours before, at least 12 hoursafter, or at the same time) administration of the cationicimmunoliposome complex.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the results of an ELISA assay showing binding ofTfRscFv-liposome-DNA complex, made by simple mixing, to DU145 cells atvarious ratios of protein/lipid and DNA/lipid.

FIG. 2 shows the results of an in vitro transfection assay usingdifferent mixing ratios of TfRscFv:lipid in DU145 cells (Luciferaseassay).

FIG. 3 shows the results of an in vitro transfection assay usingdifferent mixing ratios of TfRscFv:lipid in rat C6 cells (Luciferaseassay).

FIG. 4 shows a non-denaturing polyacrylamide gel demonstrating that >95%of the TfRscFv is bound to the liposome or liposome-p53 after simplemixing.

FIG. 5A shows the results of an XTT cytotoxicity assay showing thechemosensitivity to GEMZAR® induced in DU145 cells treated withTfRscFv-liposome-p53 prepared by simple mixing.

FIG. 5B shows the results of an XTT cytotoxicity assay showing thechemosensitivity to mitoxantrone induced in DU145 cells treated withTfRscFv-liposome-p53 prepared by simple mixing.

FIGS. 6A and 6B show the results of an XTT cytotoxicity assay showingthe chemosensitivity to GEMZAR® induced in pancreatic cancer cell lines(Colo 357 and Panc I) treated with TfRscFv-liposome-p53 prepared bysimple mixing.

FIG. 7A shows the in vitro tumor targeting ability of the systemicallyadministered TfRscFv-liposome-EGFP complex prepared by simple mixing atvarious ratios of DNA:lipid.

FIG. 7B shows the in vivo tumor targeting ability of the systemicallyadministered TfRscFv-liposome-EGFP complex prepared by simple mixing infour different tumors and using multiple batches of the TfRscFv protein.

FIG. 8 shows the effect of the combination of systemically administeredTfRscFv-liposome A-p53 prepared by simple mixing and radiation on DU145human prostate xenograft tumors.

FIG. 9 shows the results of an XTT cytotoxicity assay showingchemosensitivity to GEMZAR® induced Panc I cells treated withTfRscFv-liposome-AS HER-2 ODN.

FIG. 10A shows the effect of the combination of systemicallyadministered TfRscFv-liposome A-AS HER-2 ODN and GEMZAR® on Panc I humanpancreatic xenograft tumors.

FIG. 10B shows the effect of the combination of systemicallyadministered TfRscFv-liposome B-AS HER-2 ODN and (docetaxel) TAXOTERE®on MDA-MB-435 human breast xenograft tumors.

FIG. 11 shows the enhanced tumor imaging resulting from the systemicadministration of the TfRscFv-liposome-MAGNEVIST® complex.

FIG. 12 shows the results of an in vitro transfection assay ofsterically stabilized TfRscFv-PEG-liposome A-pLuc in MDA-MB-435 cells(Luciferase assay).

FIG. 13 shows the optimization of the molar ratio of GMC-5-193 toLipA-HoKC, and the effect of TfRscFv/LipA-HoKC/GMC-5-193 complexes atdifferent ratios of GMC-5-193 to LipA-HoKC on DU145 human prostatecancer cells.

FIG. 14 shows the effect of TfRscFv/LipA-HoKC/GMC-5-193 complexes ondifferent cell lines.

FIG. 15A shows the effect of tumor targeting liposomal delivery ofGMC-5-193 on sensitization of MDA-MB-435 human melanoma cells todoxorubicin using TfRscFv/LipA-HoKC/GMC-5-193 complexes compared to freeGMC-5-193.

FIG. 15B shows the effect of tumor targeting liposomal delivery ofGMC-5-193 on sensitization of MDA-MB-435 human melanoma cells todoxorubicin using TfRscFv/LipA/GMC-5-193 complexes compared to freeGMC-5-193.

FIG. 15C shows the effect of tumor targeting liposomal delivery ofGMC-5-193 (TfRscFv/LipA/GMC-5-193 complexes) on sensitization of B16/F10mouse melanoma cells to Cisplatin compared to free GMC-5-193, at aconcentration of 1.25 μM GMC-5-193.

FIG. 15D shows the effect of tumor targeting liposomal delivery ofGMC-5-193 (TfRscFv/LipA/GMC-5-193 complexes) on sensitization of B16/F10mouse melanoma cells to Cisplatin compared to free GMC-5-193, at aconcentration of 2 μM GMC-5-193.

FIG. 15E shows the effect of tumor targeting liposomal delivery ofGMC-5-193 (TfRscFv/LipA/GMC-5-193 complexes) on sensitization of B16/F10mouse melanoma cells to Cisplatin compared to free GMC-5-193, at aconcentration of 2.5 μM GMC-5-193.

FIG. 16A shows the effect of tumor targeting liposomal delivery ofGMC-5-193 on sensitization of normal human lung fibroblasts IMR-90 todoxorubicin using TfRscFv/LipA-HoKC/GMC-5-193 complexes.

FIG. 16B shows the effect of tumor targeting liposomal delivery ofGMC-5-193 on sensitization of normal human lung fibroblasts IMR-90 todoxorubicin using TfRscFv/LipA/GMC-5-193 complexes.

FIG. 16C shows the effect of tumor targeting liposomal delivery ofGMC-5-193 on sensitization of normal human lung fibroblasts IMR-90 tomitoxantrone using TfRscFv/LipA/GMC-5-193 complexes.

FIG. 17A shows the effect of tumor targeting liposomal delivery ofGMC-5-193 (TfRscFv/LipA-HoKC/GMC-5-193 complexes) on sensitization ofDU145 human prostate cancer cells to (docetaxel) TAXOTERE®.

FIG. 17B shows the effect of tumor targeting liposomal delivery ofGMC-5-193 (TfRscFv/LipA/GMC-5-193 complexes) on sensitization of DU145human prostate cancer cells to Mitoxantrone.

FIG. 17C shows the effect of tumor targeting liposomal delivery ofGMC-5-193 (TfRscFv/LipA/GMC-5-193 complexes) on sensitization ofMDA-MB-435 human melanoma cells to (docetaxel) TAXOTERE®.

FIG. 18 shows the effect of tumor targeting liposomal delivery ofGMC-5-193 on sensitization of B16/F10 mouse melanoma cells to CDDP.

FIG. 19A shows in vitro comparison of free GMC-5-193 orTfRscFv/LipA/GMC-5-193 complex uptake in MDA-MB-435 cells.

FIG. 19B shows in vitro comparison of free GMC-5-193 orTfRscFv/LipA-HoKC/GMC-5-193 complex uptake in MDA-MB-435 cells.

FIG. 20A shows enhanced tumor-specific uptake after systemicadministration by incorporation of a fluorescent small molecule inTfRscFv/LipA/GMC-5-193 (scL-GMC) complex.

FIG. 20B shows enhanced tumor-specific uptake after systemicadministration by incorporation of a fluorescent small molecule inTfRscFv/LipA-HoKC/GMC-5-193 complex.

FIG. 20C shows additional data demonstrating enhanced tumor-specificuptake after systemic administration by incorporation of fluorescentGMC-5-193 using the TfRscFv/LipA-HoKC/GMC-5-193 complex.

FIG. 21A shows inhibition of tumor growth in B16/F10 lung tumor bearingsyngenic mice after treatment with free GMC-5-193,TfRscFv/LipA/GMC-5-193 (scL-GMC) and TfRscFv/LipA-HoKC/GMC-5-193(scLHK-GMC) complexes.

FIG. 21B shows inhibition of tumor growth in B16/F10 lung tumor bearingsyngenic mice after treatment with the combination ofTfRscFv/LipA/GMC-5-193 (scL-GMC) and TfRscFv/LipA-HoKC/GMC-5-193(scLHK-GMC) complexes with and without Cisplatin.

FIG. 21C shows inhibition of tumor growth in B16/F10 lung tumor bearingsyngenic mice after treatment with the combination ofTfRscFv/LipA/GMC-5-193 and TfRscFv/LipA-HoKC/GMC-5-193 complexes withand without CDDP.

FIG. 22 shows cleaved caspase-3 in the serum of B16/F10 lung tumorbearing syngenic mice after treatment with the combination ofTfRscFv/LipA/GMC-5-193 and TfRscFv/LipA-HoKC/GMC-5-193 complexes withand without CDDP.

FIG. 23A shows a comparison of the effects of TfRscFv/LipA/Yk-3-250complexes at different ratios of YK-3-250 to LipA in MDA-MB-435 cells.

FIG. 23B shows a comparison of the effect of free and complexed YK-3-250on MDA-MB-435 cells.

FIG. 24A shows a comparison of the effect of Imatinib Mesylate (GLEEVEC®delivered via liposome complex and free Imatinib Mesylate (GLEEVEC® onhuman prostate cancer cells.

FIG. 24B shows a comparison of the effect of Imatinib Mesylate(GLEEVEC®) delivered via liposome complex and free Imatinib Mesylate(GLEEVEC®) on human melanoma cells.

FIG. 24C shows a comparison of the effect of Imatinib Mesylate(GLEEVEC®) on B16-F10 cells.

FIG. 25A shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (20 μM) on sensitization of MDA-MB-435 humanmelanoma cells to (docetaxel) TAXOTERE®.

FIG. 25B shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (30 μM) on sensitization of MDA-MB-435 humanmelanoma cells to (docetaxel) TAXOTERE®.

FIG. 26A shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (20 μM) on sensitization of DU145 human prostatecancer cells to Mitoxantrone.

FIG. 26B shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (30 μM) on sensitization of DU145 human prostatecancer cells to Mitoxantrone.

FIG. 27A shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (20 μM) on sensitization of PANC-1 human pancreaticcancer cells to Gemcitabine.

FIG. 27B shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (30 μM) on sensitization of PANC-1 human pancreaticcancer cells to Gemcitabine.

FIG. 28A shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (20 μM) on sensitization of B16/F10 mouse melanomacells to Cisplatin (CDDP).

FIG. 28B shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (30 μM) on sensitization of B16/F10 mouse melanomacells to Cisplatin (CDDP).

FIG. 29A shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (15 μM) on sensitization of 86/F10 mouse melanomacells to Dacarbazine (DTIC) 24 hours incubation.

FIG. 29B shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (20 μM) on sensitization of B16/F10 mouse melanomacells to Dacarbazine (DTIC) 24 hours incubation.

FIG. 29C shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (15 μM) on sensitization of B16/F10 mouse melanomacells to Dacarbazine (DTIC) 48 hours incubation.

FIG. 29D shows the effect of liposome complex delivery of ImatinibMesylate (GLEEVEC®) (20 μM) on sensitization of B16/F10 mouse melanomacells to Dacarbazine (DTIC) 48 hours incubation.

FIG. 30A shows the effect of tumor targeting liposomal delivery ofImatinib Mesylate (GLEEVEC®) (TfRscFv/LipA/Imatinib Mesylate(scL-GLEEVEC®)) on sensitization of normal human fibroblast cell lineH500 to mitoxantrone.

FIG. 30B shows the effect of tumor targeting liposomal delivery ofImatinib Mesylate (GLEEVEC®) (TfRscFv/LipA/Imatinib Mesylate(scL-GLEEVEC®)) on sensitization of normal human fibroblast cell lineH500 to (docetaxel) TAXOTERE®.

FIG. 31 shows the inhibition of B16/F10 lung tumor growth by thecombination of TfRscFv/LipA/Imatinib Mesylate (scL-GLEEVEC®) plus CDDP.

FIG. 32A shows the comparison of the effects of Erlotinib (TARCEVA®)delivered by the ligand-liposome complex (TfRscFv/LipA/Erlotinib(scL-TARCEVA®)) and free Erlotinib on human prostate cancer cells(DU145).

FIG. 32B shows the comparison of the effects of Erlotinib (TARCEVA®)delivered by the ligand-liposome complex (TfRscFv/LipA/Erlotinib(scL-TARCEVA®)) and free Erlotinib on human pancreatic cancer cells(PANC-1).

FIG. 32C shows the comparison of the effects of Erlotinib (TARCEVA®)delivered by the ligand-liposome complex (TfRscFv/LipA/Erlotinib(scL-TARCEVA®)) and free Erlotinib on human melanoma cells (MDA-MB-435).

FIG. 33A shows the effect of TfRscFv/LipA (scL) complex delivery ofErlotinib at a concentration of 3.75 uM on sensitization of humanprostate cancer cell line DU145 to mitoxantrone as compared to freeErlotinib (TARCEVA®).

FIG. 33B shows the effect of TfRscFv/LipA (scL) complex delivery ofErlotinib at a concentration of 7.5 uM on sensitization of humanprostate cancer cell line DU145 to mitoxantrone as compared to freeErlotinib (TARCEVA®).

FIG. 34A shows the effect of TtRscFv/LipA (scL) complex delivery ofErlotinib at a concentration of 3.75 uM on sensitization of humanmelanoma cells to docetaxel (TAXOTERE®) as compared to free Erlotinib(TARCEVA®).

FIG. 34B shows the effect of TfRscFv/LipA (scL) complex delivery ofErlotinib at a concentration of 7.5 uM on sensitization of humanmelanoma cells to docetaxel (TAXOTERE®) as compared to free Erlotinib(TARCEVA®).

FIG. 35 shows the effect of TfRscFv/LipA (scL) complex delivery ofErlotinib at a concentration of 7.5 uM on sensitization of humanpancreatic cancer cells PANC-1 to gemcitabine (GEMZAR®) as compared tofree Erlotinib (TARCEVA®).

FIG. 36A shows the effect of tumor targeting liposomal delivery ofErlotinib (TARCEVA®) (TfRscFv/LipA/Erlotinib (scL-TARCEVA®)) onsensitization of normal human fibroblast cell line H500 to Mitoxantrone.

FIG. 36B shows the effect of tumor targeting liposomal delivery ofErlotinib (TARCEVA®) (TfRscFv/LipA/Erlotinib (scL-TARCEVA®)) onsensitization of normal human fibroblast cell line H500 to (docetaxel)TAXOTERE®.

FIG. 36C shows the effect of tumor targeting liposomal delivery ofErlotinib (TARCEVA®) (TfRscFv/LipA/Erlotinib (scL-TARCEVA®)) onsensitization of normal human fibroblast cell line H500 to Gemcitabine(GEMZAR®).

FIG. 37 shows the effect of different ratios of Sunitinib (SUTENT®)/LipAin the TfRscFv/LipA/Sunitinib complex on DU145S human prostate cancercells as compared to free Sunitinib.

FIG. 38A shows the comparison of the effects of Sunitinib (SUTENT®)delivered by the ligand-liposome complex (TfRscFv/LipA/Sunitinib(scL-SUTENT®)) and free Sunitinib on human prostate cancer cells(DU145).

FIG. 38B shows the comparison of the effects of Sunitinib (SUTENT®)delivered by the ligand-liposome complex (TfRscFv/LipA/Sunitinib(scL-SUTENT®)) and free Sunitinib on human pancreatic cancer cells(PANC-1).

FIG. 39A shows the effect of TfRscFv/LipA (scL) complex delivery ofSunitinib at a concentration of 2.5 uM on sensitization of humanmelanoma cell line MDA-MB-435 to Docetaxel (TAXOTERE®) as compared tofree Sunitinib (SUTENT®).

FIG. 39B shows the effect of TfRscFv/LipA (scL) complex delivery ofSunitinib at a concentration of 5 uM on sensitization of human melanomacell line MDA-MB-435 to Docetaxel (TAXOTERE®) as compared to freeSunitinib (SUTENT®).

FIG. 40 shows the effect of TfRscFv/LipA (scL) complex delivery ofSunitinib at a concentration of 5 uM on sensitization of human prostatecancer cell line DU145 to mitoxantrone as compared to free Sunitinib(SUTENT®).

FIG. 41 shows the effect of TfRscFv/LipA (scL) complex delivery ofSunitinib at a concentration of 2.5 uM on sensitization of humanpancreatic cancer cell line PANC-1 to gemcitabine (GEMZAR®) as comparedto free Sunitinib (SUTENT®).

FIG. 42A shows the effect of tumor targeting liposomal delivery ofSunitinib (SUTENT®) (TfRscFv/LipA/Sunitinib (scL-SUTENT®)) onsensitization of normal human fibroblast cell line H500 to Mitoxantrone.

FIG. 42B shows the effect of tumor targeting liposomal delivery ofSunitinib (SUTENT®) (TfRscFv/LipA/Sunitinib (scL-SUTENT®)) onsensitization of normal human fibroblast cell line H500 to docetaxel(TAXOTERE®).

FIG. 42C shows the effect of tumor targeting liposomal delivery ofSunitinib (SUTENT®) (TfRscFv/LipA/Sunitinib (scL-SUTENT®)) onsensitization of normal human fibroblast cell line H500 to Gemcitabine(GEMZAR®).

FIG. 43A shows the effect of tumor targeting liposomal delivery ofSunitinib (SUTENT®) (TfRscFv/LipA/Sunitinib (scL-SUTENT®)) onsensitization of normal human lung fibroblast cell line IMR90 toMitoxantrone.

FIG. 43B shows the effect of tumor targeting liposomal delivery ofSunitinib (SUTENT®) (TfRscFv/LipA/Sunitinib (scL-SUTENT®)) onsensitization of normal human lung fibroblast cell line IMR90 todocetaxel (TAXOTERE®)).

FIG. 43C shows the effect of tumor targeting liposomal delivery ofSunitinib (SUTENT®) (TfRscFv/LipA/Sunitinib (scL-SUTENT®)) onsensitization of normal human lung fibroblast cell line IMR90 togemcitabine (GEMZAR®).

FIG. 44 shows the effect of different ratios of Gefitinib (IRESSA®)/LipAin the TfRscFv/LipA/Gefitinib complex on MDA-MB-231 human breast cancercells as compared to free Gefitinib (IRESSA®).

FIG. 45A shows the comparison of the effects of Gefitinib (IRESSA®)delivered by the ligand-liposome complex (TfRscFv/LipA/Gefitinib(scL-Gefitinib)) and free Gefitinib on human breast cancer cells(MDA-MB-231).

FIG. 45B shows the comparison of the effects of Gefitinib (IRESSA®)delivered by the ligand-liposome complex (TfRscFv/LipA/Gefitinib(scL-Gefitinib)) and free Gefitinib on human melanoma cells(MDA-MB-435).

FIG. 45C shows the comparison of the effects of Gefitinib (IRESSA®)delivered by the ligand-liposome complex (TfRscFv/LipA/Gefitinib(scL-Gefitinib)) and free Gefitinib on human prostate cancer cells(DU145).

FIG. 46A shows the effect of TtRscFv/LipA (scL) complex delivery ofGefitinib at a concentration of 12 uM on sensitization of human breastcancer cell line MDA-MB-231 to docetaxel (TAXOTERE®) as compared to freeGefitinib (IRESSA®).

FIG. 46B shows the effect of TfRscFv/LipA (scL) complex delivery ofGefitinib at a concentration of 15 uM on sensitization of human melanomacells to docetaxel (TAXOTERE®) as compared to free Gefitinib (IRESSA®).

FIG. 46C shows the effect of TfRscFv/LipA (scL) complex delivery ofGefitinib at a concentration of 8 uM on sensitization of human prostatecancer cell line (DU145) to mitoxantrone as compared to free Gefitinib(IRESSA®).

DETAILED DESCRIPTION OF THE INVENTION

Antibody- or antibody fragment-targeted cationic liposome or cationicpolymer complexes in accordance with this invention are made by a simpleand efficient non chemical conjugation method in which the components ofthe desired complex are mixed together in a defined ratio and in adefined order. Unexpectedly, the resultant complexes are as effectiveas, or more effective than, similar complexes in which the antibody orantibody fragment is chemically conjugated to the liposome or polymer.

Either a whole antibody or an antibody fragment can be used to make thecomplexes of this invention. In an exemplary embodiment, an antibodyfragment is used. Suitably, the antibody fragment is a single chain Fvfragment of an antibody. One exemplary antibody is an anti-TfRmonoclonal antibody, and a suitably antibody fragment is an scFv basedon an anti-TfR monoclonal antibody. A suitable anti-TfR monoclonalantibody is 5E9. An scFv based on this antibody contains the completeantibody binding site for the epitope of the TfR recognized by this MAbas a single polypeptide chain of approximate molecular weight 26,000. AnscFv is formed by connecting the component VH and VL variable domainsfrom the heavy and light chains, respectively, with an appropriatelydesigned peptide, which bridges the C-terminus of the first variableregion and N-terminus of the second, ordered as either VH-peptide-VL orVL-peptide-VH. Another exemplary antibody is an anti-HER-2 monoclonalantibody, and another preferred antibody fragment is an scFv based on ananti-HER-2 monoclonal antibody.

In a preferred embodiment, a cysteine moiety is added to the C-terminusof the scFv. Although not wishing to be bound by theory, it is believedthat the cysteine, which provides a free sulfhydryl group, may enhancethe formation of the complex between the antibody and the liposome. Withor without the cysteine, the protein can be expressed in E. coliinclusion bodies and then refolded to produce the antibody fragment inactive form, as described in detail in the Examples below.

Unless it is desired to use a sterically stabilized immunoliposome inthe formation of the complex, a first step in making the complexcomprises mixing a cationic liposome or combination of liposomes orsmall polymer with the antibody or antibody fragment of choice. A widevariety of cationic liposomes are useful in the preparation of thecomplexes of this invention. Published PCT application WO99/25320describes the preparation of several cationic liposomes. Examples ofdesirable liposomes include those that comprise a mixture ofdioleoyltrimethylammonium phosphate (DOTAP) anddioleoylphosphatidylethanolamine (DOPE) and/or cholesterol (chol), amixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE and/orchol. The ratio of the lipids can be varied to optimize the efficiencyof uptake of the therapeutic molecule for the specific target cell type.The liposome can comprise a mixture of one or more cationic lipids andone or more neutral or helper lipids. A desirable ratio of cationiclipid(s) to neutral or helper lipid(s) is about 1:(0.5-3), preferably1:(1-2) (molar ratio). In addition, the liposomes can also compriseendosomal disrupting peptides, such as the K[K(H)KKK]₅-K(H)KKC (HoKC)(SEQ ID NO: 2) peptide manufactured by Sigma-Genosys (The Woodlands,Tex.). The endosomal disrupting peptide HoKC may help the release ofagents in the cytoplasm of the cells.

Suitable polymers are DNA binding cationic polymers that are capable ofmediating DNA compaction and can also mediate endosome release. Apreferred polymer is polyethyleneimine. Other useful polymers includepolysine, protamine and polyamidoamine dendrimers.

The antibody or antibody fragment is one which will bind to the surfaceof the target cell, and preferably to a receptor that is differentiallyexpressed on the target cell. The antibody or antibody fragment is mixedwith the cationic liposome or polymer at room temperature and at aprotein:lipid ratio in the range of about 1:20 to about 1:40 (w:w) or aprotein polymer ratio in the range of about 0.1:1 to 10:1 (molar ratio).

The antibody or antibody fragment and the liposome or polymer areallowed to incubate at room temperature for a short period of time,typically for about 10-15 minutes, then the mixture is mixed with atherapeutic or diagnostic agent of choice. Examples of therapeuticmolecules or agents which can be complexed to the antibody and liposomeinclude genes, high molecular weight DNA (genomic DNA), plasmid DNA,antisense oligonucleotides, siRNA, peptides, ribozymes, nucleic acids,small molecules, viral particles, immunomodulating agents, proteins,imaging agents and chemical agents. Preferred therapeutic moleculesinclude genes encoding p53, Rb94 or Apoptin. RB94 is a variant of theretinoblastoma tumor suppressor gene. Apoptin is a gene that inducesapoptosis in tumor cells only. In another preferred embodiment, theagent is an antisense oligonucleotide, such as HER-2. A preferred HER-2antisense oligonucleotide has the sequence 5′-TCC ATG GTG CTC ACT-3′(Seq. ID No: 1). A third type of preferred agent is a diagnostic imagingagent, such as an MRI imaging agent, such as a Gd-DTPA agent. If theagent is DNA, such as the coding region of p53, it can be positionedunder the control of a strong constitutive promoter, such as an RSV or aCMV promoter.

The antibody or antibody fragment and liposome combination is mixed withthe therapeutic or diagnostic agent at a ratio in the range of about1:10 to 1:20 (μg of agent:nmole of total lipid) or about 1:10 to 1:40(ug of agent:nmole of total polymer) and incubated at room temperaturefor a short period of time, typically about 10 to 15 minutes. The sizeof the liposome complex is typically within the range of about 50-500 nmas measured by dynamic light scattering using a Malvern ZETASIZER® 3000.

In one embodiment of this invention, the liposome used to form thecomplex is a sterically stabilized liposome. Sterically stabilizedliposomes are liposomes into which a hydrophilic polymer, such as PEG,poly(2-ethylacrylic acid), or poly(n-isopropylacrylamide (PNIPAM) havebeen integrated. Such modified liposomes can be particularly useful whencomplexed with therapeutic or diagnostic agents, as they typically arenot cleared from the blood stream by the reticuloendothelial system asquickly as are comparable liposomes that have not been so modified. Tomake a sterically stabilized liposome complex of the present invention,the order of mixing the antibody or antibody fragment, the liposome andthe therapeutic or diagnostic agent is reversed from the order set forthabove. In a first step, a cationic liposome as described above is firstmixed with a therapeutic or diagnostic agent as described above at aratio in the range of about 1:10 to 1:20 (μg of agent:nmole of lipid).To this lipoplex is added a solution of a PEG polymer in aphysiologically acceptable buffer and the resultant solution isincubated at room temperature for a time sufficient to allow the polymerto integrate into the liposome complex. The antibody or antibodyfragment then is mixed with the stabilized liposome complex at roomtemperature and at a protein:lipid ratio in the range of about 1:5 toabout 1:30 (w:w).

The liposomal or polymer complexes prepared in accordance with thepresent invention can be formulated as a pharmacologically acceptableformulation for in vivo administration. The complexes can be combinedwith a pharmacologically compatible vehicle or carrier. The compositionscan be formulated, for example, for intravenous administration to ahuman patient to be benefited by administration of the therapeutic ordiagnostic molecule of the complex. The complexes are sizedappropriately so that they are distributed throughout the body followingi.v. administration. Alternatively, the complexes can be delivered viaother routes of administration, such as intratumoral, oral,intralesional, aerosal, percutaneous, endoscopic, topical,intraperitoneal or subcutaneous administration.

In one embodiment, compositions comprising the antibody- or antibodyfragment-targeted liposome (or polymer) and therapeutic agent complexesare administered to effect human gene therapy. The therapeutic agentcomponent of the complex comprises a therapeutic gene under the controlof an appropriate regulatory sequence. Gene therapy for various forms ofhuman cancers can be accomplished by the systemic delivery of antibodyor antibody fragment-targeted liposome or polymer complexes whichcontain a nucleic acid encoding wt p53. The complexes can specificallytarget and sensitize tumor cells, both primary and metastatic tumors, toradiation and/or chemotherapy both in vitro and in vivo.

The complexes can be optimized for target cell type through the choiceand ratio of lipids, the ratio of antibody or antibody fragment toliposome, the ratio of antibody or antibody fragment and liposome to thetherapeutic or diagnostic agent, and the choice of antibody or antibodyfragment and therapeutic or diagnostic agent.

In one embodiment, the target cells are cancer cells. Although anytissue having malignant cell growth can be a target, head and neck,breast, prostate, pancreatic, glioblastoma, renal, hepatic, cervical,lung, liposarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma,retinoblastoma, ovarian, gastric and colorectal cancers are preferredtargets.

The complexes made by the method of this invention also can be used totarget non-tumor cells for delivery of a therapeutic molecule. While anynormal cell can be a target, preferred cells are dendritic cells,endothelial cells of the blood vessels, lung cells, breast cells, bonemarrow cells, spleen cells, thymus cells, cells of the nasal passage andliver cells. Undesirable, but benign, cells can be targeted, such asbenign prostatic hyperplasia cells, over-active thyroid cells, lipomacells, and cells relating to autoimmune diseases, such as B cells thatproduce antibodies involved in arthritis, lupus, myasthenia gravis,squamous metaplasia, dysplasia and the like.

The complexes can be administered in combination with anothertherapeutic agent, such as either a radiation or chemotherapeutic agent.The therapeutic agent, or a combination of therapeutic agents, can beadministered before or subsequent to the administration of the complex,for example within about 12 hours to about 7 days. Chemotherapeuticagents include, but are not limited to, for example, doxorubicin,5-fluorouracil (5FU), cisplatin (CDDP), docetaxel, gemcitabine,pacletaxel, vinbiastine, etoposide (VP-16), camptothecia, actinomycin-D,mitoxantrone and mitomycin C. Radiation therapies include gammaradiation, X-rays, UV irradiation, microwaves, electronic emissions andthe like.

Diagnostic agents also can be delivered to targeted cells via theliposome or polymer complexes. Agents which can be detected in vivofollowing administration can be used. Exemplary diagnostic agentsinclude electron dense materials, magnetic resonance imaging agents andradiopharmaceuticals. Radionuclides useful for imaging includeradioisotopes of copper, gallium, indium, rhenium, and technetium,including isotopes ⁶⁴Cu, ⁶⁷Cu, ¹¹¹In, ^(99m)Tc, ⁶⁷Ga or ⁶⁸Ga. Imagingagents disclosed by Low et al. in U.S. Pat. No. 5,688,488, incorporatedherein by reference, are useful in the present invention.

The complexes made in accordance with the method of this invention canbe provided in the form of kits for use in the systemic delivery of atherapeutic molecule by the complex. Suitable kits can comprise, inseparate, suitable containers, the liposome, the antibody or antibodyfragment, and the therapeutic or diagnostic agent. The components can bemixed under sterile conditions in the appropriate order and administeredto a patient within a reasonable period of time, generally from about 30minutes to about 24 hours, after preparation. The kit componentspreferably are provided as solutions or as dried powders. Componentsprovided in solution form preferably are formulated in sterilewater-for-injection, along with appropriate buffers, osmolarity controlagents, etc.

In a further embodiment, the present invention provides liposomalcomplexes wherein the diagnostic and/or therapeutic agents are one ormore small molecules encapsulated within the interior of the liposome,contained within the hydrocarbon chain region of the bilayer,complexed/associated with the inner and/or outer monolayer (e.g., viastatic interaction or chemical/covalent interaction), or a combinationof any or all of these possibilities. As used herein, the term “smallmolecule” refers to a low molecular-weight pharmaceutical, therapeuticand/or diagnostic agent (examples of the latter being markers, dyes,etc.), that generally has a molecular weight of less than about 10 kD,suitably less than about 5000 Daltons, and more suitably less than about1000 Daltons, for example about 100 to about 900 Daltons, about 200 toabout 800 Daltons, about 300 to about 700 Daltons, about 400 to about600 Daltons, or about 500 Daltons, as well as salts, esters, and otherpharmaceutically acceptable forms of such compounds.

Examples of small molecules include compounds useful for treatingpatients that are suffering from or pre-disposed to any disease state,including, but not limited to, cancers (e.g., a breast cancer, a uterinecancer, an ovarian cancer, a prostate cancer, a testicular cancer, alung cancer, a leukemia, a lymphoma, a colon cancer, a gastrointestinalcancer, a pancreatic cancer, a bladder cancer, a kidney cancer, a bonecancer, a neurological cancer, a head and neck cancer, a skin cancer, asarcoma, an adenoma, a carcinoma and a myeloma); infectious diseases(e.g., bacterial diseases, fungal diseases, parasitic diseases and viraldiseases (such as a viral hepatitis, a disease caused by a cardiotropicvirus; HIV/AIDS, flu, SARS, and the like)); and genetic disorders (e.g.,anemia, neutropenia, thrombocytopenia, hemophilia, dwarfism and severecombined immunodeficiency disease (“SCID”); autoimmune disorders (e.g.,psoriasis, systemic lupus erythematosus and rheumatoid arthritis) andneurodegenerative disorders (e.g., various forms and stages of multiplesclerosis, Creutzfeldt-Jakob Disease, Alzheimer's Disease, and thelike).

Exemplary small molecules useful for treatment of cancers (i.e.anticancer small molecules) include, but are not limited to smallmolecules that inhibit tubulin polymerization, anti-angiogenic smallmolecules, kinase inhibitors, and the like. Small molecules for use inthe present invention suitably have a pKa of about 2 to about 9, and inmany cases, will have several pKas (i.e., 2, 3, 4, etc) within thisrange. Small molecules for use in the practice of the present inventioncan be water-soluble, slightly water-soluble, or poorly water soluble(including compounds that are not soluble in water).

In suitable embodiments, the small molecules for use in the practice ofthe present invention include, but are not limited to tubulinpolymerization inhibitors, such as GMC-5-193 (and analogs thereof) andYK-3-250 (and analogs thereof).

GMC-5-193 (and analogs thereof) is a thalidomide analog which hasantimicrotubule and anti-angiogenic effects in several cancer celllines. GMC-5-193 inhibits human cancer cell proliferation withantiproliferative activities. The potency of this molecule is similar tothat of vincristine, a well-known antimitotic agent. Several studiessuggest that the antiproliferative effect may be due to inhibition oftubulin polymerization in several types of cancer cells. These analogsinitiated mitotic accumulation and formation of abnormal mitoticspindles in cancer cells.

From the x-ray structure, it has been confirmed that GMC-5-193 docksright into the area of greatest amino acid difference near the taxol andcolchicines binding sites in βIII human tubulin. However, in examiningin vivo efficacy, the poor water solubility of this small moleculehinders its administration. Solubility is an important consideration interms of systemic drug bioavailability, because insolubility furtherlimits drug efficacy and the subsequent need for increased dosagecompromises patient tolerance. To circumvent this problem, atumor-targeting liposomal delivery system for this molecule, usingtumor-targeted ligand (TfRscFv) and cationic liposome conjugated with(or without) HoKC, a synthetic pH-sensitive histidylated oligolysine wasutilized. HoKC was included in the complex to improve the anticancereffect of the targeting complex, designed to aid in endosomal escape.The drug is expected to diffuse into the cytoplasm from where it istransferred to the nucleus exerting its cytotoxic effects or releasedinto the extracellular compartment, where it can have cytotoxic effectson other tumor cells (bystander effect).

Additional small molecules for use in the practice of the presentinvention include tyrosine kinase inhibitors, such as, but not limitedto:

Additional small molecules for use in the practice of the presentinvention include pharmaceutical compounds, as well as marker dyes andother molecules for diagnosis. Examples of such small molecules are wellknown and easily identified by those skilled in the art, and many can befound on databases such as Pharmabase (National Center for ResearchSources, National Institutes of Health). General classes ofpharmaceutical small molecules that can be used in the practice of thepresent invention include, but are not limited to, compounds involved inregulating membrane transport (e.g., channels, pumps, receptors,transporters); compounds involved in metabolism (such as ATP inhibitors,electron transport controllers, inhibitors of amino acid or fatty acidsynthesis, ceramide analogs, etc.); intracellular messengers (e.g.,kinase inhibitors, etc); compounds involved in regulating cellsignaling; compounds involved in regulating cellular area; as well asother well known classes of small molecules. Additional examples ofsmall molecule classes and compounds can be found throughout U.S. Pat.Nos. 7,041,651, 7,033,775, 7,005,255 and 6,900,198, the disclosures ofeach of which are incorporated by reference herein in their entireties.

As described herein, small molecules are suitably encapsulated,contained or complexed/associated with the liposome complexes of thepresent invention by simply mixing the one or more small molecules withthe liposomes during processing. Suitable ratios of smallmolecule:liposome complexes are readily determined by the ordinarilyskilled artisan. For example, the molar ratio of small molecules toliposome complex is suitably in the range of about 0.2:7 to about 14:7(small molecule:liposome), suitably at a molar ratio of about 1:7 toabout 12:7, about 1:7 to about 10:7, about 2:7 to about 9:7, about 4:7to about 8:7, about 5:7 to about 8:7, about 2.8:7 or about 7:7 (smallmolecule:liposome). As described throughout, examples of desirablecationic liposomes for delivery of small molecules include those thatcomprise a mixture of dioleoyltrimethylammonium phosphate (DOTAP) anddioleoylphosphatidylethanolamine (DOPE) and/or cholesterol (chol), amixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE and/orchol. The ratio of the lipids can be varied to optimize the efficiencyof uptake of the therapeutic molecule for the specific target cell type.The liposome can comprise a mixture of one or more cationic lipids andone or more neutral or helper lipids. A desirable ratio of cationiclipid(s) to neutral or helper lipid(s) is about 1:(0.5-3), preferablyabout 1:(1-2) (molar ratio). Examples of ratios of various lipidsinclude, but are not limited to:

LipA DOTAP/DOPE 1:1 molar ratio LipB DDAB/DOPE 1:1 molar ratio LipCDDAB/DOPE 1:2 molar ratio LipD DOTAP/Chol 1:1 molar ratio LipE DDAB/Chol1:1 molar ratio LipG DOTAP/DOPE/Chol 2:1:1 molar ratio LipHDDAB/DOPE/Chol 2:1:1 molar ratio (DOTAP = dioleoyltrimethylaminnoniumphosphate, DDAB = dimethyldioctadecylammonium bromide; DOPE =dioleoylphosphatidylethanolamine; chol = cholesterol).

In one embodiment, the present invention provides methods of preparingsmall molecule-comprising antibody- or antibody fragment-targetedcationic immunoliposome complexes comprising preparing an antibody orantibody fragment; mixing the antibody or antibody fragment with acationic liposome to form a cationic immunoliposome, wherein theantibody or antibody fragment is not chemically conjugated to thecationic liposome; and mixing the cationic immunoliposome with a smallmolecule to form said antibody- or antibody fragment-targeted-cationicimmunoliposome complex. While not chemically conjugated to the cationicliposome, the antibody or antibody fragment directlyassociates/complexes with the liposome, e.g., via a charge-charge, orother non-chemical conjugation interaction, to form the immunoliposomes.

In suitable embodiments, the antibody fragment is a single chain Fvfragment, for example, an anti-transferrin receptor single chain Fv(TfRscFv). Examples of suitable lipids for use in preparing the smallmolecule-comprising cationic immunoliposomes are described herein, andinclude, mixtures of dioleoyltrimethylammonium phosphate withdioleoylphosphatidylethanolamine and/or cholesterol; and mixtures ofdimethyldioctadecylammonium bromide withdioleoylphosphatidylethanolamine and/or cholesterol. Suitably, thecationic immunoliposome is mixed with the small molecule at a molarratio in the range of about 0.2:7 to about 14:7 (smallmolecule:immunoliposome), suitably at a molar ratio of about 1:7 toabout 12:7, about 1:7 to about 10:7, about 2:7 to about 9:7, about 4:7to about 8:7, about 5:7 to about 8:7 or about 7:7 (smallmolecule:immunoliposome). Exemplary small molecules for use in thepractice of the present invention include those described herein, aswell as additional small molecules known in the art and readilyidentifiable by the ordinarily skilled artisan. Suitably, the smallmolecules are anticancer small molecules, such as, but not limited to,GMC-5-193, YK-3-250, imatinib mesylate, erlotinib hydrochloride,sunitinib malate, gefitinib and analogs and derivatives thereof, as wellas others described herein and/or that will be familiar to theordinarily skilled artisan. In a further embodiment, the presentinvention provides small molecule-comprising cationic immunoliposomecomplexes prepared by the methods described herein.

In a further embodiment, the present invention provides antibody- orantibody fragment-targeted cationic immunoliposome complexes comprisinga cationic liposome, an antibody or antibody fragment, and a smallmolecule, wherein the antibody or antibody fragment is not chemicallyconjugated to the cationic liposome. The small molecule(s) can beencapsulated within the cationic liposome, contained with a hydrocarbonchain region of the cationic liposome, associated with an inner or outermonolayer of the cationic liposome (including the head-group region), orany combination thereof. Suitably, the cationic immunoliposomes of thepresent invention are unilamellar liposomes (i.e. a single bilayer),though multilamellar liposomes which comprise several concentricbilayers can also be used. Single bilayer cationic immunoliposomes ofthe present invention comprise an interior aqueous volume in whichagents (e.g., small molecules) can be encapsulated (suitablywater-soluble agents). They also comprise a single bilayer which has ahydrocarbon chain region (i.e., the lipid chain region of the lipids) inwhich agents (e.g., small molecules) can be contained (suitablylipid-soluble agents). In addition, agents (e.g., small molecules) canbe complexed or associated with either, or both, the inner monolayerand/or the outer monolayer of the liposome membrane (i.e., the headgroupregion of the lipids). In further embodiments, agents (e.g., smallmolecules) can be encapsulated/associated/complexed in any or all ofthese regions of the cationic immunoliposome complexes of the presentinvention.

In a still further embodiment, the present invention provides methods oftreating a patient suffering from, or pre-disposed to, a disease state,comprising administering the small molecule-comprising cationicimmunoliposome complexes of the present invention to the patient. Theimmunoliposome complexes can be administered via any desired route,including, but not limited to, intravenous, oral, topical, viainhalation, intramuscular injection, intratumoral injection,intralesional injection, aerosal, percutaneous, endoscopic, topical,intraperitoneal, or subcutaneous administration or other injectionroutes. As used herein, the term patient includes both animal patients(e.g., mammals such as dogs, cats, pigs, sheep, etc) as well as humans.

Suitably, the methods of the present invention are used to treatpatients suffering from, or predisposed to, cancer. In furtherembodiments, the methods of treating patients suffering from, orpredisposed to, cancer can further comprise administering achemotherapeutic agent to the patient in addition to the administrationof the small molecule-comprising immunoliposome complex. In suitableembodiments, the methods of the present invention comprise administeringan immunoliposome complex comprising a small molecule selected from thegroup consisting of GMC-5-193, YK-3-250, imatinib mesylate, erlotinibhydrochloride, sunitinib malate, gefitinib and analogs and derivativesthereof, along with a chemotherapeutic agent selected from the groupconsisting of doxorubicin, cisplatin, mitoxantrone, (docetaxel)TAXOTERE® and CDDP. The small molecule-comprising immunoliposome complexand the chemotherapeutic agent can be administered at the same time, orcan be administered at different times (e.g., before or after oneanother). Suitably, the chemotherapeutic agent is administered before orafter the small molecule-comprising immunoliposome complex, (e.g., atleast 6 hours before or after, at least 12 hours before or after, atleast 24 hours before or after, at least 48 hours before or after, etc.,administration of the cationic immunoliposome complex). In furtherembodiments, the chemotherapeutic agent and the smallmolecule-comprising immunoliposome complex are administered at the sametime to the patient. Appropriate dosages and timings or administrationof the small molecule-comprising immunoliposome complexes and thechemotherapeutic agents are easily determined by those of skill in theart, based on information contained herein and that is readily availablein the art.

In a further embodiment, the present invention provides methods ofenhancing the effectiveness of a chemotherapeutic agent comprisingadministering a cationic immunoliposome complex of the present invention(e.g., a cationic immunoliposome complex comprising a small molecule) inconjunction with the chemotherapeutic agent to a patient. Suitable smallmolecules and chemotherapeutic agents include those described throughoutas well as those known in the art. The small molecule-comprisingimmunoliposome complex and the chemotherapeutic agent can beadministered at the same time, or can be administered at differenttimes. Suitably, the chemotherapeutic agent is administered before orafter the small molecule-comprising immunoliposome complex, (e.g., atleast 6 hours before or after, at least 12 hours before or after, atleast 24 hours before or after, at least 48 hours before or after, etc.,administration of the cationic immunoliposome complex). In furtherembodiments, the chemotherapeutic agent and the smallmolecule-comprising immunoliposome complex are administered at the sametime to the patient.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

Example 1 Construction and Purification of TfRscFv with a 3′-Cysteine

Plasmid expression vector pDFH2T-vecOK was obtained from Dr. DavidFitzgerald, NCI. This vector encodes the single chain fragment for the5E9 antibody, which recognizes the human transferrin receptor (CD71).The VH-linker-Vκ TfRscFv was obtained by PCR amplification of thedesired fragment. A cysteine moiety was added at the 3′ end of theTfRscFv protein. Two forms of this vector were constructed. The firstcontains a pelB leader signal sequence, for transport to the periplasmicspace, and a His Tag. The presence of the His Tag aids in detection ofthe protein, thus simplifying development of the purification protocol.Although this form was used for the initial testing, FDA guidelinesrecommend that no extraneous sequences be present for use in clinicaltrials. Therefore, a second form minus both of these sequences also wasmade.

Using PCR amplification the nucleotide sequence for the cysteine residueand a NotI restriction site were introduced at the 3′ end. Similarly, a5′ NcoI site also was incorporated. The PCR product was cloned into NcoIand NotI sites of the commercial vector pET26b(+) (Novagen) thusproducing a protein product containing both the pelB leader signalsequence and the His Tag. Growth in bacterial culture containing IPTGyielded an approximate 100 fold increase in single chain proteinexpression which was maximum at approximately 10 hours of IPTGinduction. This protein was found primarily in the insoluble fraction(inclusion bodies).

The above construct also was modified to eliminate both the His Tag andpelB sequences in the final protein product. To accomplish this, thepET26b(+) vector was cut at the Nde I enzyme site 5′ of the pelBsequence. PCR amplification inserted an Nde I site at the 5′ end of theVH-linker-Vκ scFv for the TfR sequence. In addition to the nucleotidesequence for the cysteine residue and the Nod restriction site at the 3′end, a DNA stop codon was introduced adjacent to the cysteine sequenceand before the NotI site. The PCR product was cloned into the NdeI andNotI sites of commercial expression vector pET26b(+) (Novogen). Thus,the protein product of this construct will not contain either the pelBsequence or the His-tag.

The majority of the cys-TfRscFv protein (approximately 90%) was foundnot to be soluble but to be contained within inclusion bodies.Therefore, the protein from the constructs described above was isolatedfrom the inclusion bodies by sonication, treatment with 6 Mguanidine-HCl, 200 mM NaCl (6 M GuHCl buffer) and purified via SephacrylS-200 gel filtration column chromatography. Refolding of the cys-TfRscFvprotein was accomplished by dialysis at 4° C. against decreasingconcentrations of guanidine-HCl. Alternatively, the cys-TfRscFv proteinwas prepared by isolation of the inclusion bodies by sonication withTriton X-100 followed by solubilization in 6 M Guanidine-HCl, 0.1 μMtris-HCl pH-8.0, 2 mM EDTA pH=8.0 and dithioerythritol. Refolding wasaccomplished by mixing with a buffer composed of 0.1 M Tris-HCl pH=8.0,0.5 M L-arginine-HCl, 2 mM EDTA and 0.9 mM glutathionine and holding at4° C. for 36-48 hours, followed by dialysis at 4° C. for 20-24 hoursagainst 20 mM Tris-HCl (pH=9.0), 100 mM Urea, and 2 mM EDTA (pH=8.0).After dialysis, the cys-TfRscFv was purified by ion exchangechromatography with Q-sepharose, followed by concentration (an usingAmicon ultrafiltration device) and dialysis at 4° C. for 30 hoursagainst PBS (pH=7.4) plus 0.06 M sodium chloride. After purification,SDS-PAGE showed a single band of the solubilzed, refolded cys-TfRscFvprotein with the correct molecular weight of approximately 28-30 kDa (asdescribed in WO 00/50008). The cys-TfRscFv protein is stored at −80° C.

Example 2 Preparation of Cys-TfRscFv-Liposome by Simple Mixing

Published PCT application WO 99/25320, incorporated herein by reference,describes the preparation of several cationic liposomes. The cationicliposomes prepared are clear solutions, their compositions and ratiosare as follows:

LipA DOTAP/DOPE 1:1 molar ratio LipB DDAB/DOPE 1:1 molar ratio LipCDDAB/DOPE 1:2 molar ratio LipD DOTAP/Chol 1:1 molar ratio LipE DDAB/Chol1:1 molar ratio LipG DOTAP/DOPE/Chol 2:1:1 molar ratio LipHDDAB/DOPE/Chol 2:1:1 molar ratio (DOTAP = dioleoyltrimethylaminnoniumphosphate, DDAB = dimethyldioctadecylammonium bromide; DOPE =dioleoylphosphatidylethanolamine; chol = cholesterol)

It is well known by those knowledgeable in the field that conjugatedTfRscFv-immunoliposome retains its immunologic activity. We haveestablished that the cys-TfRscFv can be chemically conjugated tolipoplex (PCT application WO 00/50008) and can efficiently transfecthuman prostate tumor cells in vitro and in vivo. It is common practicefor single chain antibody fragments to be attached to liposomes usingvarious chemical conjugation methods. We performed studies to determineif a simple mixing of the cys-TfRscFv and the cationic liposome (whichdoes not contain any lipid with a reducible group such as Maleimide DOPEor any reducible group), instead of chemical conjugation, would resultin formation of an immunologically active complex that could stillefficiently bind to and transfect tumor cells. A series ofcys-TfRscFv-immunoliposome complexes was prepared by mixing thecys-TfRscFv with liposome A at defined ratios of single chain protein toliposome ranging from 1/25 to 1/36 (w/w). Based upon the ELISA data withthe conjugated cys-TfRscFv complex the ratio of DNA to nmoles totallipid in the mixed complex also was varied from 1/8 to 1/18. Thepreparation of the complexes was in accordance with the followinggeneral procedure: The appropriate amount of 2 mM liposome (A-Hdescribed above) is mixed with any water required to give a desiredvolume and inverted to mix. To the liposome-water the appropriate amountof cys-TfRscFv is added to give the desired ratio and mixed by gentleinversion 5-10 seconds. This mixture is kept at room temperature for 10minutes (again inverted gently for 5-10 seconds after approximately 5minutes). At the same time, the appropriate amount of DNA is mixed byinversion for 5-10 seconds with any water required to give a desiredvolume. Typically, for use in an in vitro assay, it is desirable thatthe concentration of DNA is in the range of about 0.01 μg to about 2 μgper well; for in vivo use, it is desirable to provide about 5 μg toabout 100 μg of DNA per injection. The DNA solution is quickly added tothe cys-TfRscFv-liposome solution and the mixture is inverted for 5-10seconds. The final mixture is kept at room temperature for 10 minutes,gently inverting again for 5-10 seconds after approximately 5 minutes.For use in vivo 50% dextrose or 50% sucrose is added to a finalconcentration of 5-10% (V:V) and mixed by gentle inversion for 5-10seconds. A specific example at a preferred ratio of 1:30(cys-TfRscFv:liposome, w:w) and 1:14 (μg DNA:n mole total Lipid) is asfollows: For 40 μg of DNA in a final volume of 800 μl mix 183 μl waterwith 280 μl of 2 mM liposome solution. Add 34 μl of cys-TfRscFv (with aconcentration of 0.4 μg/ml). Mix 183 μl water with 40 μl of 1 μg/1 μlDNA. Add 80 μl of 50% Dextrose as the last step.

The size of the final complex prepared by the method of this inventionis between 100 and 400 (number value) with a zeta potential of between25 and 35 as determined by dynamic light scattering using a MalvernZetasizer 3000. This size is small enough to efficiently pass throughthe tumor capillary bed and reach the tumor cells.

An ELISA assay to assess the binding ability of the mixed complex tohuman prostate cancer DU145 cells was performed. For comparison, thecomplexes made with the conjugated immunoliposome were also included inthe assay. The results shown in FIG. 1 clearly demonstrate that theimmunoliposome complex prepared by simple mixing of the cys-TfRscFvprotein with the cationic liposome binds to DU145 cells at least as wellas those prepared through conjugation. Similar to the conjugatedcomplex, a ratio of 1/30 protein to lipid and 1/14 DNA to lipid wasfound to have the highest binding ability. As was also previouslyobserved with the conjugated complexes, the binding decreased in a DNAdose dependent manner. These findings indicate that simple mixing ofcomponents can form a complex that retains its immunologic activity.Identical optimal ratios were found in human prostate DU145 cells, andRAT C6 cells using the Luciferase assay (FIGS. 2 and 3) and in humanpancreatic cancer cell line Panc I (Table I, II) using enhanced greenfluorescence protein (EGFP) to assess the transfection efficiency.

TABLE I Transfection Efficiency of cys-TfRscFv-Liposome A in Panc ICells Prepared by Simple Mixing Assessed Using the EGFP Reporter Gene IRatio DNA:Total Lipids (μg:nmoles) % Fluorescent Cells 1:8  20 1:10 221:12 35 1:14 50 1:16 24 1:18 20 The ratio of cys-TfRscFv:Liposome was1:3 (w:w)

TABLE II Transfection Efficiency of cys-TfRscFv-Liposome A in Panc ICells Prepared by Simple Mixing Assessed Using the EGFP Reporter Gene IIRatio cys-TfRscFv:Lipids (w:w) % Fluorescent Cells 1:26 14 1:28 14 1:3030 1:32 28 1:34 15 1:36 18

To establish the efficiency of the binding of the cys-TfRscFv to theliposome complex by simple mixing, a non-denaturing polyacrylamide gelwas used. Mixed cys-TfRscFv-liposome A-p53 complex andcys-TfRscFv-Liposome A without p53 DNA were loaded on the gel along withfree cys-TfRscFv in amounts equal to ⅕ or 1/10 the amount of cys-TfRscFvused to prepare the complexes. The complexes were prepared using theratio of cys-TfRscFv:liposome of 1:30 (w:w) and DNA:total lipid of 1:14(μg:n mol total lipid). The free cys-TfRscFv complexes serve asquantitation standards, since under non-denaturing conditions thecomplex can not enter the gel, only free, unbound cys-TfRscFv canmigrate into it. After transferring to membrane, the gel was probed withan anti-cys-TfRscFv antibody using the ECL Western Blot detection kit(Amersham). Comparison of the low signal level for the two complexes(with and without p53 DNA) shown in FIG. 4 with the signals from thefree cys-TfRscFv standards indicates that greater than 95% of thecys-TfRscFv is incorporated into the complex by simple mixing of thecomponents.

Example 3 In Vitro Chemosensitization of Human Cancer Cell Lines byCys-TfRscFv-Immunoliposome Delivered Wtp53

Experiments were performed to determine how effective thecys-TfRscFv-Liposome-p53 complex prepared by simple mixing would be insensitizing prostate tumor cells to the drugs GEMZAR® (gemcitabine HCl;manufactured by Eli Lilly and Co.) and NOVANTRONE® (mitoxantrone,Immunex Corp.) both of which currently are used for the treatment ofprostate cancer. The prostate tumor cell line DU145, which harborsmutant p53, was employed in these studies. The XTT cytotoxicity assay(66) was used to establish the level of chemosensitivity induced by thecys-TfRscFv-Liposome-p53 complex of this invention. 5×10³ DU145 cellswere plated/well of a 96 well plate. After 24 hours, the cells weretransfected with the mixed cys-TfRscFv-Liposome-p53 complex. Thecys-TfRscFv-Liposome-p53 complex was prepared by mixing at a ratio of1:30 (w:w) (cys-TtRscFv:Liposome A) and 1:14 (μg p53 DNA: nmoles totallipid). One day after transfection, anti-neoplastic agents were added atincreasing concentrations (in triplicate). The XTT assay was performedapproximately 3 days later and IC₅₀ values, the drug concentrationyielding 50% growth inhibition, calculated. As shown in FIG. 5A,treatment with the cys-TfRscFv-Liposome-p53 complex increased thesensitivity of the cells to GEMZAR® by 8-fold. For FIG. 5A, the IC₅₀Values (nM) are as follows: cys-TfRscFv-LipA-p53: 0.5; cys-TfRscFv-LipA:4.0; cys-TfRscFv-LipA-Vec: 4.0; Untransfected: 5.0. The foldsensitization for Vec vs p53=8 and for UT vs p53=10.

Similarly, DU145 cells were sensitized to the drug mitoxantrone by17.5-fold (FIG. 5B). For FIG. 5B, the IC₅₀ values (ng/ml) were asfollows: cys-TfRscFv-LipA-p53: 0.08; cys-TfRscV-LipA: 1.20;cys-TfRscFv-LipA-Vec: 1.40 and Untransfected: 1.80. The foldsensitization for Vec vs p53=17.5 and for UT vs p53=22.5. Similarstudies were performed using human pancreatic cancer cell line Panc I.4×10³ Panc I cells per well were plated, and the XTT assay performed asabove. A preferred ratio of 1:30 (cys-TfRscFv:liposome A w:w) and 1:14(μg p53 DNA: nmoles total lipid) also was used here. As with DU145 therewas significant sensitization of the tumor cells to chemotherapeuticagents (FIGS. 6A and B). At a p53 DNA concentration of 0.06 μg/wellthere was a 23.8 fold increase in sensitization to GEMZAR® using themixed cys-TfRscFv-liposome DNA complex (FIG. 6A). For FIG. 6A, the IC50values were as follows: cys-TfRscFvLipA-p53: 0.21 nM;cys-TfRscFvLipA-Vec: 5.00 nM and TfLipA-p53: 0.30 nM. The IC50 ofcys-TfRscFvLipA-Vec/IC50 of cys-TfRscFrLipA-p53=23.8. No sensitizationwas observed when empty vector in place of p53 was used. There wasdramatic increase in response of the Panc I cells at a p53 DNAconcentration of 0.08 μg DNA/well (FIG. 6B). Here an almost 200 foldincrease in sensitization was observed. For FIG. 6B, the IC50 valueswere as follows: cys-TfRscFvLipA-p53: 1.8 nM; cys-TfRscFvLipA-Vec: 350nM; and cys-TfRscFvLipA: 600 nM. The IC₅₀ of cys-TfRscFvLipA-Vec/IC50 ofcys-TfRscFvLipA-p53=194.44. Therefore, these in vitro studiesdemonstrate that the cys-TfRscFv-liposome, prepared by simple mixing,can efficiently transfect wtp53 into prostate tumor cells and sensitizethem to conventional chemotherapeutic agents.

Example 4 In Vivo Tumor Targeting by the Cys-TfRscFv-LipA-EGFP Preparedby Simple Mixing

DU145 tumors were subcutaneously induced in female athymic nude (NCRnu/nu) mice. Mice were I.V. tail vein injected three times over a 24hour period with cys-TfRscFv-LipA-EGFP (enhanced green fluorescenceprotein) (TfRscFvII) prepared by simple mixing at a scFv:Liposome ratioof 1/30 but at various DNA:total lipid ratios (1/10, 1/11, 1/12, 1/13,1/14) at 32 ug DNA/injection. For comparison, a complex at 1/30, 1/14made via the conjugation method (TfRscFv III in FIG. 7B) and a differentbatch of single chain at 1/30, 1/14 (TfRscFv I in FIG. 7B) also wereinjected into mice. 60 hours post injection the mice were sacrificed,tumor and lung harvested and protein isolated for Western Blot Analysisusing an anti-EGFP antibody. Unliganded LipA-EGFP complex (UL),Tf-LipA-EGFP complex (Tf) and BSA-LipA-EGFP complex (BSA) were injectedinto mice as controls. FIG. 7A. As shown in the DU145 tumor an EGFP bandis observed in the positive controls Tf, TfRscFvII, and in TfRscFvI.More significantly, a strong EGFP signal was found in TfRscFvII at theDNA to lipid ratio of 1/14. In contrast, only very low level of EGFPexpression was evident in normal lung tissue. Therefore, thecys-TfRscFv-Lipoplex prepared by simple mixing can target tumoreffectively after systemic administration.

To assess the reproducibility of the mixing, different batches ofcys-TfRscFv (1 to V) were complexed to Liposome A-EGFP by simple mixingat the preferred ratio of 1:30 (scFv:liposome w:w) and 1:14 (μgDNA:nmoles total lipid). Human prostate DU145, bladder HTB-9, breastMDA-MB-435 and head and neck JSQ-3 xenograft tumors were subcutaneouslyinduced as above. The complexes also were I.V. tail vein injected threetimes over a 24 hour period. Tf-LipA-EGFP(Tf) and unliganded LipA-EGFPcomplex (UL) were used as controls. 60 hours after injection the micewere sacrificed and the tumor and liver were harvested and analyzed asabove. Targeting is evident with all of the mixed complexes in the fourtumor types (FIG. 7B). However, there is almost no signal in normaltissue (liver). The identical membrane was probed for Actin levels toshow equal loading.

Example 5 Radio/Chemosensitization of Human Xenograft Tumors bySystemically Administered Cys-TfRscFv-Liposome-p53 Prepared by SimpleMixing

Efficacy studies were performed to further confirm the ability of thecys-TfRscFv-immunoliposome complex of this invention to bind and deliverwtp53 efficiently to tumor cells in vivo. Mice bearing subcutaneousDU145 tumors of approximately 60-90 mm³ were injected, via the tailvein, three times a week (a total of 10 injections) withcys-TfRscFv-Liposome-p53. This complex was prepared by simple mixing ata ratio of 1/30 (cys-TfRscFv:Liposome A, w:w) and 1/14 (μg DNA/nmolestotal lipid). The tumor area was selectively exposed to 2.0 Gy dailyfractionated doses of γ-radiation to a total of 32 Gy (FIG. 8). Theanimals treated with the mixed cys-TFRscFv-liposome A complex plusradiation had significant tumor growth inhibition. Similar findings alsowere observed using the combination of the anticancer drug Gemzar® andthe cys-TFRscFv-immunoliposome of this invention delivering tumorsuppressor gene Rb94 to a human bladder carcinoma xenograft tumor(HTB-9), and in Panc I xenografts treated with GEMZAR® andcys-TFRscFv-Liposome carrying either another gene inducing apoptosis(Apoptin) or p53.

These findings demonstrate that a complex made by the method of thisinvention can comprise a variety of genes (incorporated into plasmidvectors) for effective delivery in vive to cancer cells as a therapeutictreatment.

Example 6 Chemosensitization of Pancreatic Cancer Cells In Vitro byAntisense HER-2 Oligonucleotides Delivered by Cys-TFRscFv-Liposome aPrepared by Simple Mixing

This example demonstrates the usefulness of this invention inefficiently delivering molecules other than genes to tumor cells fortherapeutic treatment. The complex was prepared as in Example 2,however, the DNA encapsulated here was an 18 mer phosphorothioatedoligonucleotide (ODN) directed against the initiation codon of the HER-2gene (AS HER-2) (51). The ratio used was as above for plasmid DNA 1:30(cys-TfRscFv:liposome, w:w) and 1:14 (nmoles ODN:n mole total lipids).Panc I cells, at 4×10³ cells/well, were seeded in a 96 well plate. Thecells were transfected 24 hours later by cys-TfRscFv-LipA-AS HER-2prepared by the method of this invention. Tf-LipA-AS HER-2 andcys-TfRscFv-LipA-SC ODN were used as controls. SC ODN is a scrambled ODNthat has the same nucleotide composition as the AS HER-2 ODN but inrandom order. As shown in FIG. 9 the cys-TfRscFv-Lip A-AS HER-2 complexprepared by the method of this invention was able to sensitizepancreatic cancer cell line Panc I to the effects of chemotherapeuticagent GEMZAR® by over 11 fold. This increase in sensitization isidentical to that resulting from transfection with the positive controlTf-LipA-AS HER2 complex. For FIG. 9, the IC₅₀ values were as follows:TfRscFv-LipA3-AS-HER-2: 16 nM; Tf-LipAe-AS-HER-2: 14 nM andTfR-scFv-LipAe-SC: 200 nM. The IC₅₀ of TfR-scFv-LipAe-SC/IC₅₀ ofTfR-scFv-LipAe-AS-HER-2—12.5.

Example 7 In Vivo Chemosensitization of Human Xenograft Tumors bySystemically Delivered Cys-TFRscFv-LipA-AS HER-2 ODN Prepared by SimpleMixing

In this example, the ability of the cys-TfRscFv liposome-DNA complexprepared by the method of this invention to deliver an antisensemolecule to tumor cells in vivo after systemic delivery is demonstrated.To show the universality of this delivery system two different humanxenograft mouse tumor models (pancreatic cancer and breast cancer) wereemployed. In the first (FIG. 10A) Panc I subcutaneous xenograft tumorswere induced in female athymic nude (NCR nu/nu) mice. When the tumorswere 100-200 mm³ in size the animals were injected with thechemotherapeutic agent GEMZAR® (intraperitoneally) and withcys-TfRscFv-LipA AS HER-2 prepared by the method of this invention(I.V.). The complex was made using the ratio of 1:30(cys-TfRscFv:liposome, w:w) and 1:15 (n mole ODN:n mole total lipid). Inaddition to the I.V. injections the complex described above also wasintratumorally injected. One group of animals received GEMZAR® only anda second control group received GEMZAR® plus the complex carrying emptyvector. Treatment with GEMZAR® alone was not able to significantlyinhibit pancreatic tumor growth. In contrast (FIG. 10A), the combinationof GEMZAR® and AS-HER-2 ODN delivered by the cys-TfRscFv-Lip A complexprepared by the method of this invention not only significantlyinhibited tumor growth but also resulted in tumor regression.

Significant tumor growth inhibition of human breast cancer xenografttumors also was observed with the combination of the drug TAXOTERE®(docetaxel; manufactured by Aventis Pharmaceuticals, Collegeville, Pa.)and I.V. administered cys-TfRscFv-LipB AS HER-2 prepared by the methodof this invention (FIG. 10B). While liposome formulation B was used inthe breast tumor, the same ratios as described above for Panc I wereemployed.

Example 8 Enhancement of MRI Image by Delivery of Imaging AgentMAGNEVIST® by Cys-TFRscFv-Liposome a Prepared by Simple Mixing

This example demonstrates the ability to encapsulate MRI imaging agentsand form a cys-TfRscFv-Liposome-imaging agent complex by the method ofthis invention. The complex prepared by the method of this invention canbe administered intravenously resulting in increased enhancement of thetumor image for both primary tumor and metastases. These imaging agentscan include, but are not limited to, MAGNEVIST® (Gd-DTPA) (Schering AG).The ratios used to form the complex by simple mixing are the preferredratios of 1:30 (cys-TfRscFv:liposome, w:w) and 1:14 (ug imagingagent:nmoles lipid). In these studies 16 ul of MAGNEVIST® were used inthe complex.

FIG. 11 shows the results from one I.V. injection of thecys-TfRscFv-LipA-MAGNEVIST® made by the method of this invention intomice bearing subcutaneous xenograft tumors of human head and neck (toppanel), breast (middle panel) or prostate (bottom panel) origin. Ahigher level of imaging agent enhancement is evident in the tumor thatreceived the cys-TfRscFv-LipA-MAGNEVIST® as compared to that receivingfree MAGNEVIST® demonstrating the benefit of administering the imagingagent using the complex prepared by the method of this invention. Inother experiments an increased uptake in the tumor as compared to thesurrounding normal tissue also was observed.

Similar enhancement also was observed using syngenetic mouse lungmetastasis model. B₁₆/F₁₀ mouse melanoma cells were injectedintravenously into C57BL/6 mice. These cells form tumor nodules in themouse lungs. The cys-TfRscFv-Liposome-MAGNEVIST® complex was prepared bythe method of this invention also using the preferred ratios of 1:30 and1:14. The complex was I.V. administered and the tumor modules imaged viaMRI. Compared to free MAGNEVIST®, the encapsulated imaging agent alsohas a prolonged uptake in the tumor since the peak enhancement with thecomplex is later than that of the free MAGNEVIST®.

Example 9 Preparation of Sterically Stabilized Immunoliposomes by SimpleMixing

Liposomal complexes are rapidly cleared from the blood stream by thereticuloendothelial system. In an effort to prolong this circulationtime sterically stabilized liposomes have been formulated that have ahydrophilic polymer such as PEG integrated into the liposome complex.Various methods have been devised to include a targeting ligand such asan antibody or antibody fragment in the PEG-liposome complex. Most, ifnot all, of these methodologies involve a chemical conjugation step tolink the antibody or antibody fragment to the PEG. Such harsh chemicalreactions and the method used to form the complex can result in loss ormasking of antibody activity. In this example, we demonstrate that thecys-TfRscFv protein can be linked to a PEG-liposome molecule by simplemixing and that the resultant complex can more efficiently transfecthuman tumor cells.

To form this complex, a lipoplex consisting of one of the cationic lipidformulations given in Example 2 was mixed with nucleic acid at a ratioof 1:14 (ug DNA:nmoles lipid) as described in Example 2. To thislipoplex was added the commercially available NHS-PEG-MAL polymer (2%)in 25 mM HEPE Buffer (pH 7.2). The solution was gently inverted for 3-5seconds and incubated at room temperature for 1.5 hours. To form thecys-TfRscFv-PEG-Liposome-DNA complex, the cys-TfRscFv protein was addedto the PEG-lipoplex at a ratio of 1:8 (cys-TfRscFv:liposome, w:w),inverted gently and kept at room temperature for 10 minutes to 1 hour,then used to transfect the cells in vitro. Other ratios in the range of1:5 to 1:30 (cys-TfRscFv:liposome, w:w) could also be employed to formthe complex. For in vivo use, 50% Dextrose was added to a finalconcentration of 5% after the incubation, mixed gently by inversion andinjected into animals. Alternatively, the final complex could have beenstored at 4° C. overnight (12-18 hr).

In the experiment shown here the nucleic acid was pLuc, a plasmid DNAthat codes for the firefly luciferase gene. Human melanoma cellsMDA-MB-435 were plated at 5×10⁴ cells/well. Twenty-four hours later theywere transfected with the cys-TfRscFv-PEG-LipA-pLuc as described inExample 3 and the transfection efficiency assessed by the level ofluciferase activity. As shown in FIG. 12 the cys-TfRscFv-PEG-LipA-pLuccomplex prepared by the method of this invention was able to transfectthe target cells with better efficiency than the PEG-LipA-pLuc withoutthe targeting cys-TfRscF protein.

Thus the method of simple mixing described here also can be used as asimple, non-destructive means of preparing sterically stabilizedtargeted immunoliposomes.

Example 10 Preparation and Characterization of Small Molecule(GMC-5-193)-Comprising-Immunoliposomes by Simple Mixing

To improve the in vitro and in vivo anticancer effects of the smallmolecule GMC-5-193, a tumor-targeting liposomal complex comprising thesmall molecule was prepared. In addition to a TfRscFv/LipA complex, acationic liposome conjugated with endosomal disrupting peptide(LipA-HoKC) was also prepared. The endosomal disrupting peptide HoKC mayhelp the release of GMC-5-193 in the cytoplasm of the cells to affecttubulin polymerization in cytoplasm. The ligand-liposome complexpreferentially targets tumor cells due to elevated levels of thecorresponding receptor on their surface. High levels of expression ofthe ligand-liposome delivered gene were evident in primary tumors andmetastasis, but not in normal tissue such as liver, lung, bone marrow,and intestinal crypts. In this Example, a liposome complex of thepresent invention, comprising the transferrin receptor single chain(TfRscFv), was used to deliver GMC-5-193 to cancer cells in vitro and invivo to evaluate the in vitro and in vivo bio-efficacy of the lipoplexcomprising GMC-5-193.

Materials and Methods

1,2-Dioeoyl-3-trimethylammonium propane (DOTAP), dioleolylphosphatidylethanolamine (DOPE), and N-maleimido-phenylbutyrate DOPE (MPB-DOPE) werepurchased from Avanti Polar Lipids (Alabaster, Ala.). TheK[K(H)KKK]₅-K(H)KKC (HoKC) (SEQ ID NO: 2) peptide was manufactured bySigma-Genosys (The Woodlands, Tex.).

Synthesis of GMC-5-193

The compound GMC-5-193 was synthesized at the department of Chemistry,University of Virginia. It has a molecular weight of 359.4 Da and itsstructure was confirmed by mass spectrometry and NMR. The pKa values ofamide proton and amine proton were 15 and 9, respectively. 2.5 mg/mLstock solution of the compound was prepared in DMSO.

Cell Lines and Culture

The human prostate cancer cell line DU145 (HTB-81) and mouse melanomacell line B16/F10 (CRL-6475) were obtained from the American TypeCulture Collection (ATCC; Manassas, Va.). DU145 was cultured in Eagleminimum essential medium with Earls salts (EMEM) supplemented with 10%heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 50μg/mL each of penicillin, streptomycin, and neomycin. B16/F10 (ATCC,CRL-6475) was cultured in Dulbecco modified Eagle medium (DMEM)supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 50μg/mL each of penicillin, streptomycin, and neomycin. The human melanomacell line MDA-MB-435, human breast cancer cell line MDA-MB-231, andhuman pancreatic cancer cell line PANC-1 were cultured in improved MEM(IMEM) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and50 μg/mL each of penicillin, streptomycin, and neomycin. The metastaticcell line MDA435/LCC6, was developed from MDA-MB-435 ascites.MDA435/LCC6 was cultured in IMEM supplemented with 5% heat-inactivatedFBS, 2 mM L-glutamine, and 50 μg/mL each of penicillin, streptomycin,and neomycin. Normal human lung fibroblast IMR-90 cells, a gift from Dr.I. Panyutin (Nuclear Medicine Department, National Institutes of Health,Bethesda, Md.), were cultured in EMEM supplemented with 10%heat-inactivated FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids,1 mM sodium pyruvate, and 50 μg/mL each of penicillin, streptomycin, andneomycin. Normal (non-cancerous) skin fibroblast cell line H500 wascultured in EMEM supplemented with 1 mM Sodium pryuvate, 1 mMNon-essential amino acids plus 10% heat inactivated fetal bovine serum,2 mM L-glutamine and 50 g/mL each of penicillin, streptomycin, andneomycin. EMEM was purchased from MediaTech (Herndon, Va.) and the othercell culture media and ingredients were obtained from Biofluids(Rockville, Md.).

Preparation of TfRscFv/LipA/GMC-5-193 Complexes

Cationic liposomal formulation LipA (DOTAP:DOPE or DDAB:DOPE at a 1:1 to1:2 molar ratio) were prepared using the ethanol injection method asdescribed throughout, and in U.S. patent application Ser. No.09/914,046, the disclosure of which is incorporated by reference hereinin its entirety. TfRscFv/LipA/GMC-5-193 complexes were prepared asfollows. After 10 minutes incubation with rotation or stirring at roomtemperature of a mixture of LipA and TfRscFv (ratio of TfRscFv to LipA,1:1 to 1:40 (wt/wt), more suitably 1:10 to 1:30 wt/wt), the GMC-5-193 atthe appropriate concentration was added mixed by inversion or stirringat room temperature and incubated for 10 minutes. For animal injection,dextrose or sucrose was added to each sample to a final concentration of1% to 20%, more suitably 5-10%. The molar ratio of GMC-5-193 to Liposomewas from 0.2:7 to 14:7, more suitably 2:7 to 8:7, most suitably 7:7 or2.8:7. The sizes of the complexes were determined by dynamic lightscattering at 25° C. with a ZETASIZER® 3000HS system (Malvern, UnitedKingdom).

Preparation of TfRscFv/LipA-HoKC/GMC-5-193 Complexes

Cationic liposomal formulations for use with the HoKC peptide andLipA-MPB (DOTAP:DOPE:MPB-DOPE or DDAB:DOPE:MPB-DOPE at a 1:1:0.1 to1:2:0.1 molar ratios) were prepared using the ethanol injection method.The LipA-HoKC liposome was then prepared using the coupling reactionbetween the cationic liposomes carrying the maleimide group and thepeptide-carrying terminal cysteine group as previously described. Yu,W., et al., “Enhanced transfection efficiency of a systemicallydelivered tumor-targeting immunolipoplex by inclusion of a pH-sensitivehistidylated oligolysine peptide.” Nucleic Acids Research 32:e48 (2004).An aliquot of 0.1 mmol of the peptide with a free thiol group oncysteine was added to 2 mmol of LipA-MPB in 10 mM HEPES (pH 7.4)solution and rotated at room temperature for 2 hours. The resultingLipA-HoKC had a lipid concentration of 1.4 mM.TfRscFv/LipA-HoKC/GMC-5-193 complexes were prepared as follows. After 10minutes incubation with rotation or stirring at room temperature of amixture of LipA-HoKC and TfRscFv (ratio of TfRscFv to LipA-HoKC, 1:1 to1:40 (wt/wt), more suitably 1:10 to 1:30 (wt/wt)), the GMC-5-193 at theappropriate concentration was added mixed by inversion or stirring atroom temperature and incubated for 10 minutes. For animal injection,dextrose or sucrose was added to each sample to a final concentration of1% to 20%, more suitably 5-10%. The molar ratio of GMC-5-193 to Liposomewas from 0.2:7 to 14:7, more suitably 2:7 to 8:7, most suitably 7:7 or2.8:7. The sizes of the complexes were determined by dynamic lightscattering at 25° C. with a ZETASIZER® 3000HS system (Malvern, UnitedKingdom).

In Vitro Cell Viability and Optimization of the TfRscFv/LipA/GMC-5-193or TfRscFv/Liposome-HoKC/GMC-5-193 Complexes

For in vitro cytotoxicity studies, 5 to 5.5×10³ cells/well in 100 μL ofthe appropriate growth medium of each cell line were plated in a 96-wellplate. After 24 hours, the cells were washed with serum-free medium,overlaid with 100 μL of TfRscFv/Liposome/GMC-5-193 complexes(TfRscFv/LipA/GMC-5-193 or TfRscFv/LipA-HoKC/GMC-5-193 as appropriate)or free GMC-5-193 in serum-free medium in increasing concentrations,incubated for 4-6 hours, suitably 5 hours, and then supplemented withFBS. The cells were then incubated for an additional 24-72 hours,suitably 48 hours at 37° C. in a humidified atmosphere containing 5%CO₂. Afterward, the wells were washed with IMEM without phenol red andthe cell-viability XTT-based assay was performed according to themanufacturer's protocol (Boehringer Mannheim, Indianapolis, Ind.). Inthe presence of an electron-coupling reagent, XTT, sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonate is converted into orange formazan by dehydrogenase in themitochondria of living cells. The formazan absorbance, which correlatesto the number of living cells, was measured at 450 nm using a microplatereader (Molecular Devices, Menlo Park, Calif.). The IC₅₀ yielding 50%growth inhibition was interpolated from the graph of the log of drugconcentration versus the fraction of surviving cells.

In Vitro Chemosensitization

For the chemosensitization study, 4-5×10³ cells/well in 100 μL wereseeded in a 96-well plate. After 24 hours, the cells were washed withserum-free medium, overlaid with 100 μL of TfRscFv/Liposome/GMC-5-193complexes (TfRscFv/LipA/GMC-5-193 or TfRscFv/LipA-HoKC/GMC-5-193 asappropriate) or free GMC-5-193 at 1.25-2.5 μM as GMC-5-193, incubatedfor 4-6 hours, suitably 5 hours, and then FBS was added to each well.The cells were incubated for an additional 24-72 hours, suitably 19hours, followed by the addition of the appropriate supplemented mediumwith or without chemotherapeutics in increasing concentrations, andincubation continued for approximately 24-72 hours, suitably 48 hours.The chemotherapeutic drugs used were doxorubicin (Bedford Labs, Bedford,Ohio), docetaxel (TAXOTERE®; Aventis Pharmaceuticals, Bridgewater,N.J.), mitoxantrone (NOVANTRONE®, Immunex Corp., Seattle Wash.) andcisplatin (CDDP; Bedford Labs, Bedford, Ohio). The XTT assays wereperformed to assess the degree of sensitization to thechemotherapeutics, and IC₅₀ values of each cell were calculated. Foldsensitization equals the following: IC₅₀ untransfected/IC₅₀ eachcomplex.

In Vitro Confocal Imaging

For the confocal imaging, 5.0×10⁴ cells/well of MDA-MB-435 were seededon the glass in a 24-well plate and, after 24 hours, washed withserum-free medium, treated with TfRscFv/Liposome/GMC-5-193 complexes(TfRscFv/LipA/GMC-5-193 or TfRscFv/LipA-HoKC/GMC-5-193 as appropriate)or free OMC-5-193, and incubated for 6 hours. Six hours after treatment,the cells were washed with phosphate-buffered saline (PBS) twice, fixedwith 4% paraformaldehyde in PBS, and washed again with PBS. Then, thecells were mounted on the slide glass, using PROLONG® Antifade Kit(Molecular Probes, Eugene, Oreg.). For nuclear staining, DAPI,blue-fluorescent counterstain reagent in Select FX Nuclear Labeling Kitfor fixed cells (Molecular Probes) was used according to themanufacturer's protocol. For imaging, an Olympus FLUOVIEW®-300 laserscanning confocal system located in the GUMC Microscopy and ImagingShared Resource (MISR) was used.

In Vivo Tumor Targeting

Human metastatic cells MDA435/LCC6 (8×10⁶) suspended in PBS wereinjected intravenously into the tail vein of athymic nude mice. The micecarrying MDA-MB-435/LCC6 xenograft tumors were injected intravenouslywith free GMC-5-193, TfRscFv/LipA/GMC-5-193, TfRscFv/LipA-HoKC/GMC-5-193or LipA/GMC-5-193 (unliganded complex) at 9 mg/kg GMC-5-193 per mouse.This is a molar ratio of 2.8:7 (small molecule to liposome). The ratioof single-chain antibody fragment to liposome in each complex was 1:30(w:w). Three hours after injection, the liver, lung and any othervisible tumor, were excised and examined under a fluorescence microscope(Nikon SMZ-1500 EPI-Fluorescence stereoscope system).

In Vivo Efficacy Studies

Mouse melanoma cells B16/F10 (1×10⁵) suspended in PBS were injectedintravenously into the tail vein of C57BL/6 mice. The mice carryingB16/F10 tumors were intravenously injected with, free GMC-5-193 only, orTfRscFv/Liposome/GMC-5-193 complexes (TfRscFv/LipA/GMC-5-193 orTfRscFv/LipA-HoKC/GMC-5-193 as appropriate) alone or in combination withCDDP at a dose of 3 mg/kg GMC-5-193/injection two to three times a weekto at total of 7 injections. The molar ratio of GMC-5-193 to LipA orLipA-HoKC in each complex was 7 to 7. The ratio of single-chain antibodyfragment to liposome in each complex was 1:30 (w:w). Certain groups alsoreceived CDDP only, given as twice weekly injections to a total of 6injections. The first two CDDP injections were at 2.5 mg/kg. Allsubsequent injections were at 2 mg/kg. After 3 weeks of treatment, themice were sacrificed, the lungs were excised, and the blood from eachmouse was withdrawn. The organs were fixed in 10% formaldehyde andpreserved in 70% ethanol before being photographed.

Western Blot Analysis of Cleaved Caspase-3 from Mice Serum

The blood collected from the retroorbital sinus of mice was centrifugedat 1,000 rpm for 10 minutes at room temperature after coagulation atroom temperature for 1 hr. The serum was transferred to a new tube andcentrifuged again at 10,000 g for 20 minutes. The supernatant serum waspurified using a P-30 Micro-Bio-Spin Chromatography Column (BIORAD,Hercules, Calif.). Fifteen microliters of the fractions was loaded on a4% to 12% gradient NuPAGE gel (Invitrogen Life Technologies, Carlsbad,Calif.) and run until the 10 kDa protein marker has run off the gel(approximately 1.5 to 2 hours). After electrophoresis, the proteins weretransferred onto a Protran BA 85 nitrocellulose transfer membrane(Schleicher and Schuell, BioScience, Keene, N. H.). Finally, a Westernblot analysis for cleaved caspase-3 was performed with cleaved caspase-3(17 kD) antibody. To block nonspecific binding, the membrane wasincubated at room temperature for 1 hour with 5% nonfat dry milk in 10mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl and 0.05% Tween 20(TBST). The blot was probed with cleaved caspase-3 rabbit primaryantibody (Cell Signaling Technology, Beverly, Mass.) at 1:1000 dilution,in 5% milk TEST, overnight at 4° C. and then washed three times (15minutes each) with TBST. The protein was detected using goat antirabbitsecondary antibody (Jackson ImmunoResearch Laboratories, West Grove,Pa.), ECL Western blotting detection reagent, and Hyperfilm ECL(Amersham, Piscataway, N.J.).

Results

In Vitro Optimization of the Molar Ratio of TfRscFv/LipA-HoKC/GMC-5-193Complex

The TfRscFv/LipA-HoKC/GMC-5-193 complex was prepared as detailed aboveand the molar ratio of the small molecule to liposome of theTfRscFv/LipA-HoKC/GMC-5-193 complex was optimized. The cytotoxic effectof complexed GMC-5-193 at different ratios of GMC-5-193 to LipA-HoKC onDU145 human prostate cancer cells was examined. 5.5×10³ cells/well wereseeded in a 96-well plate and treated after 24 hours withTfRscFv/LipA-HoKC/GMC-5-193 complexes or free GMC-5-193. The XTT assayswere performed 48 hours after treatment to assess cytotoxicity, and theIC₅₀ values (the drug concentration yielding 50% growth inhibition) werecalculated from the concentration-cell viability curve. FIG. 13 showsthe IC₅₀ values of DU145 cells treated with each complex. The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). At the range of the molar ratio of GMC-5-193 to liposome of2:7˜7:7, a decrease in the IC₅₀ values were observed in cells treatedwith the complexed GMC-5-193 in comparison with cells treated with freeGMC-5-193, reducing the IC₅₀ values from 7.7±0.9 μM to 2.2±5 μM. At themolar ratio of GMC-5-193 to liposome of 8:7. The IC₅₀ values of cellstransfected with either complexed or free GMC-5-193 were similar. TheGMC-5-193 complex at the molar ratio of GMC-5-193 to liposome of 7:7 waschosen for further experiment because there was no significantdifference in the IC₅₀ values of DU145 cells treated with the complexedGMC-5-193 at the range of the molar ratio of GMC-5-193 to liposome of2:7˜7:7. The particle size of the complexed GMC-5-193 at the molar ratioof GMC-5-193 to liposome of 7:7 (i.e. 1:1) was about 300 nm in 5%dextrose.

The Effect of Free or Complexed GMC-5-193 on Cell Kill (Sensitivity) inDifferent Cell Lines

FIG. 14 shows the comparison of the level of cell kill of the complexedGMC-5-193 and free GMC-5-193 in different cell lines (DU145, MDA-MB-435,MDA435/LCC6, B16/F10. Here 4˜5.5×10³ cells/well were seeded in a 96-wellplate and after 24 hours, treated with TfRscFv/LipA-HoKC/GMC-5-193complexes, free GMC-5-193. The concentrations of GMC-5-193 were 50nM˜31.25 μM. The molar ratio of GMC-5-193 to LipA-HoKC in the complexeswas 1:1. The ratio of single-chain antibody fragment to liposome in eachcomplex was 1:30 (w:w). The XTT assays were performed 48 hours aftertreatment to assess cytotoxicity and the IC₅₀ values were calculated.The IC₅₀ values of DU145, MDA435/LCC6 and B16/F10 cells treated with theTfRscFv/LipA-HoKC/GMC-5-193 were lower than those treated with freeGMC-5-193, indicating increased sensitivity to this molecule. MDA-MB-435showed similar sensitivity to the TfRscFv/LipA-HoKC/GMC-5-193 complex orFree GMC-5-193. The greatest difference in sensitivity between free andcomplexed GMC-5-193 was seen with DU145 cells (reducing the IC₅₀ valuefrom 7.9 uM with free to 3.2 uM with complex) and with B16/F10 cells(reducing the IC₅₀ value range from 6.6±0.8 μM to 4.2±0.0 μM).

Table III below shows the results of similar experiments performedcomparing free GMC-5-193 and the TfRscFv/LipA/GMC-5-193 complex.

TABLE III IC₅₀ values (μM)* obtained with XTT viability assay Cell linesFree GMC-5-193 TfRscFv/LipA/GMC-5-193 DU145 11.3 ± 0.6 3.9 ± 0.5MDA-MB-435  4.0 ± 0.5 2.5 ± 0.1 B16-F10 >31 5.7 ± 0.4 *Mean of threeseries of measurements ± S.D.Thus, complexing GMC-5-193 with the TfRscFv/LipA-HoKC of this inventionincreases the response of multiple human and mouse cell lines to thesmall molecule, as compared to the response to the free small moleculeas is currently used in the art.In Vitro Chemosensitization by the TfRscFv/Liposome/GMC-5-193 Complex

GMC-5-193 has microtubule disruptive and anti-angiogenic effects. Theeffect of combination therapy of free and ligand/liposome complexedGMC-5-193 with conventional chemotherapeutics was studied in order todetermine if the anticancer effect of the conventional chemotherapiescould be enhanced and to compare the level of enhancement of thecomplexed small molecule as compared to free small molecule. The abilityof the ligand/liposome/GMC-5-193 (TfRscFv/LipA-HoKC/GMC-5-193) complexto sensitize MDA-MB-435 human melanoma cells to doxorubicin was examinedand the results are presented in FIG. 15A. Here, 4.5×10³ cells/well wereseeded in a 96-well plate and after 24 hours, treated withTfRscFv/LipA-HoKC/GMC-5-193 complex, free GMC-5-193 or LipA-HoKC only.The concentration of GMC-5-193 was 1.25 μM per well. The molar ratio ofGMC-5-193 to LipA-HoKC in the complexes was 1:1. The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). After 24 hours, doxorubicin was added in concentrationsincreasing from 2.07 to ˜2070 ng/mL. After 48 hours, XTT assays wereperformed to assess cell viability in response to treatments. Each pointin FIG. 15A represents the mean of triplicate samples±standarddeviation. IC₅₀ values represent the concentration of drug resulting in50% growth inhibition. UT=untransfected. The anthracycline antibioticdoxorubicin was chosen because it is one of the most common of thechemotherapeutics and has been used for combination therapy with themicrotubule-targeted, tubulin-polymerizing agents for breast cancer.IC₅₀ values were calculated. Cells transfected with LipA-HoKC withoutGMC-5-193 showed an IC₅₀ similar to that of untransfected cells,indicating that the liposomes themselves are nontoxic. Cells treatedwith free GMC-5-193 exhibited a slightly reduced, but not significantlydifferent, IC₅₀, indicating that free GMC-5-193 does not effectivelyinhibit cell growth. However, a significant (˜50-fold) increase insensitization was observed in cells treated with the complexed GMC-5-193in comparison with cells treated with free GMC-5-193, with the IC₅₀value declining from 300 to 8 ng/mL.

The results of a similar study examining the effects of doxorubicinonly, doxorubicin plus free GMC-5-193 (GMC) and doxorubicin plusTfRscFv/LipA/GMC-5-193 (scL-GMC) complexes (i.e. cationicimmunoliposomes without the HoKC peptide) is shown in FIG. 15B. Themolar ratio of GMC-5-193 to LipA in the complexes was 1:1. The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). A ˜10 fold increase in sensitization was observed in cellstreated with the complexed GMC-5-193 in comparison to both cells treatedwith doxorubicin only and doxorubicin plus free GMC-5-193.

A study of the effects of increasing doses of GMC-5-193, administeredeither free or as TfRscFv/LipA/GMC-5-193 complex, on sensitization onB16/F10 cells to cisplatin (CDDP) was performed and is shown in FIG.15C-E. The concentration of GMC-5-193 was 1.25, 2 and 2.5 uM,respectively. The molar ratio of GMC-5-193 to LipA in the complexes was1:1. The ratio of single-chain antibody fragment to liposome in eachcomplex was 1:30 (w:w). In FIG. 15C the concentration of GMC-5-193 was1.25 μM per well and the XTT assay was incubated for 9 hours. In FIG.15D the concentration of GMC-5-193 was 2 μM per well and the XTT assaywas incubated for 7 hours. In FIG. 15E the concentration of GMC-5-193was 2.5 μM per well and the XTT assay was incubated for 9 hours. At thelowest dose of GMC-5-193 neither the free nor complexed GMC-5-193enhances the response to CDDP (FIG. 15C). However, the degree ofsensitization increases as the doses of GMC-5-193 increases to 2 uM and2.5 uM. In both cases the complexed small molecule has an increasedeffect on the cells as compared to CDDP only and free GMC-5-193. Thisdifference is most pronounced at the 2 uM dose. Thus, under thistreatment protocol, an increase in sensitization was observed in cellstreated with the complexed GMC-5-193 in comparison to cells treated withfree GMC-5-193 at the same concentration, illustrating the beneficialeffects of delivery using the complexes. Thus, there is a dose-dependentincrease in sensitization with the complex delivered GMC-5-193.

The above series of experiments also demonstrate that an increasedresponse is obtained when either TfRscFv/LipA-HoKC or TtRscFv/LipA areused to deliver the small molecule.

The specificity of the effect of the small molecule when included aspart of the complex of this invention is shown if FIG. 16A-C with normallung fibroblast cell line IMR-90. The above sensitization results withB16/F10 and MDA-MB-435 were dramatically different from the results withnormal human lung fibroblasts IMR-90 under the same conditions. 4.5×10³cells/well were seeded in a 96-well plate and after 24 hours treatedwith TfRscFv/LipA-HoKC/GMC-5-193 complex, free GMC-5-193 or LipA-HoKConly (FIG. 16A). The concentration of GMC-5-193 was 1.25 μM per well.The molar ratio of GMC-5-193 to LipA-HoKC in the complexes was 1:1. Theratio of single-chain antibody fragment to liposome in each complex was1:30 (w:w). After 24 hours, doxorubicin was added in increasingconcentrations from 2.07 ng/mL to ˜2070 ng/mL. After 48 hours, XTTassays were performed to assess cell viability in response totreatments. Each point represents the mean of triplicatesamples±standard deviation. IC₅₀ values represent concentration of drugresulting in 50% growth inhibition. UT=untransfected. In IMR-90 cells,the fold sensitizations of LipA-HoKC only, free GMC-5-193, and complexedGMC-5-193 were not statistically different, showing little or no effectof GMC-5-193 on normal cells whether administered alone or complexedwith the TfRscFv/Lip-HoKC. This is further demonstrated since in IMR-90the IC₅₀ with complexed GMC-5-193 was 200 ng/mL compared with 8 ng/mL inMDA-MB-435 cells, representing a 25-fold difference between the normaland tumor cell lines. In comparison, after treatment with freeGMC-5-193, the difference of the IC₅₀ values between the two cell lineswas only 1.1-fold. Thus GMC-5-193 is specifically more effective ontumor cells when delivered as a component of the TfRscFv/LipA-HoKCcomplex than when administered as free GMC-5-193.

Results of a similar experiment examining the effect of TfRscFV/LipA(without HoKC) delivery of GMC-5-193 on sensitization of normal humanlung fibroblasts IMR-90 to doxorubicin are shown in FIG. 16B. Again,administration of the small molecule with the complex did not sensitizethe normal cells to the chemotherapeutic agent.

Similarly in a study with IMR-90 and a different chemotherapeutic agentthe effect of tumor targeting liposomal delivery of GMC-5-193(TfRscFv/LipA/GMC-5-193) on sensitization to mitoxantrone was examined(FIG. 16C). Here also complexing the small molecule did not effect theresponse of normal cells to mitoxantrone.

The effect of the complex on sensitization of another human tumor cellline, DU145 human prostate cancer cells, to (docetaxel) TAXOTERE® wasalso examined (FIG. 17A). 4.5×10³ cells/well were seeded in a 96-wellplate and after 24 hours treated with TfRscFv/LipA-HoKC/GMC-5-193complexes. The concentration of GMC-5-193 was 1.25 μM per well. Themolar ratio of GMC-5-193 to LipA-HoKC in the complexes was 1:1. Theratio of single-chain antibody fragment to liposome in each complex was1:30 (w:w). After 24 hours, (docetaxel) TAXOTERE® was added inincreasing concentrations from 0.08 nM to ˜82.7 nM. After 48 hours XTTassays were performed to assess cell viability in response totreatments. Each point represents the mean of triplicatesamples±standard deviation. IC₅₀ values represent the concentration ofdrug resulting in 50% growth inhibition. UT-untransfected. (docetaxel)TAXOTERE® is a microtubule-targeted, tubulin-polymerizing agent that hasbeen demonstrated to exert a high level of clinical activity. This drugwas chosen because it is one of the first-line chemotherapeutics used incombination therapy for prostate cancer. The fold sensitizations ofDU14S cells to (docetaxel) TAXOTERE® after treatment with LipA-HoKConly, free GMC-5-193, and TfRscFv/LipA-HoKC/GMC-5-193 were 1.1, 1.2, and47.1, respectively, indicating that the level of response to (docetaxel)TAXOTERE® was highly enhanced in cells treated with complexed GMC-5-193.

The results of a similar study with a different chemotherapeutic agent,comparing the degree of sensitization to mitoxantrone by free GMC-5-193,LipA only and TfRscFv/LipA/GMC-5-193 complexes in DU145 human prostatecancer cells is shown in FIG. 17B. The molar ratio of GMC-5-193 to LipAin the complexes was 1:1. The ratio of single-chain antibody fragment toliposome in each complex was 1:30 (w:w). A ˜3 fold increase insensitization was observed in cells treated with the complexed GMC-5-193in comparison to cells treated with mitoxantrone only, whereas only a1.7 fold increase was seen with free GMC-5-193. The fold sensitizationis doubled when the complex is used.

The results of an additional study comparing the degree of sensitizationto (docetaxel) TAXOTERE® by free GMC-5-193 (GMC), LipA only andTfRscFv/LipA/GMC-5-193 complexes in MDA-MB-435 human melanoma cells isshown in FIG. 17C. The molar ratio of GMC-5-193 to LipA in the complexeswas 1:1. The ratio of single-chain antibody fragment to liposome in eachcomplex was 1:30 (w:w). A ˜10 fold increase in sensitization wasobserved in cells treated with the complexed GMC-5-193 in comparison tocells treated with (docetaxel) TAXOTERE® only, double that with freeGMC-5-193.

Next, the comparison of the effect of treatment with the complexedGMC-5-193 and CDDP in the mouse syngeneic lung metastasis model wasexamined. FIG. 18 shows the effect of the complex on sensitization ofB16/F10 cells to CDDP as compared to free GMC-5-193. 3.5×10³ cells/wellwere seeded in a 96-well plate and transfected after 24 hours withLipA-HoKC only, free GMC-5-193 and TfRscFv/LipA-HoKC/GMC-5-193 complex.The concentration of GMC-5-193 was 2 μM per well. The molar ratio ofGMC-5-193 to LipA-HoKC in complex was 1:1. The ratio of single-chainantibody fragment to liposome in each complex was 1:30 (w:w). After 24hours, CDDP was added in increasing concentrations from 0.4 μM-200 μM.The XTT assays were performed 48 hours after CDDP treatment to assessthe degree of sensitization to CDDP. Each point is the mean oftriplicate samples±standard deviation. IC₅₀ values are the drugconcentration yielding 50% growth inhibition. UT=untransfected. Anincrease in sensitization by the complexed GMC-5-193 over free GMC-5-193was observed, reducing the ICs value from 42 to 20 μM. The foldsensitization of the complex was 2.6, once again double that of freeGMC-5-193. Thus, the results from all of the above in vitro experimentssupport the observation that GMC-5-193 complexed with the targetedliposome, both TfRscFv/LipA and TfRScFv/LipA-HoKC, sensitizes cancercells more effectively to conventional chemotherapeutics than whendelivered as free GMC-5-193 as is currently used in the art.

In Vitro Confocal Imaging

Confocal imaging was used to compare the internalization of theGMC-5-193 when delivered as free GMC-5-193 and when complexed with thetargeted liposomes (with and without HoKC peptide), taking advantage ofthe inherent fluorescence of GMC-5-193. MDA-MB-435 cells were exposedfor 6 hours to either free GMC-5-193 or complexed GMC-5-193, then washedwith PBS, fixed with 4% paraformaldehyde in PBS, and visualized byconfocal microscopy. FIG. 19A shows comparison between uptake of freeGMC-5-193 and that delivered to the cells by the TfRscFv/LipA/GMC-5-193complex; FIG. 19B shows comparison between uptake of free GMC-5-193 andthat delivered by TfRscFv/LipA-HoKC/GMC-5-193 (scLHK-GMC) complex. 5×10⁴cells/well were seeded on glass slides in a 24-well plate and after 24hours treated with TfRscFv/Liposome/GMC-5-193 complex or free GMC-5-193.The molar ratio of GMC-5-193 to LipA or LipA-HoKC in the complexes was1:1. The ratio of single-chain antibody fragment to liposome in eachcomplex was 1:30 (w:w). Six hours after treatment, cells were washedwith phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde inPBS, and mounted on the glass slides. The Olympus FLUOVIEW®-300 laserscanning confocal system was used to visualize fluorescence.Significantly increased fluorescence uptake by the cells was observedwhen the small molecule GMC-5-193 was complexed with either theTfRScFv/LipA or the TfRScFv/LipA-HoKC complexes of this invention whencompared to cells treated with free GMC-5-193.

The GMC-5-193 fluorescent distribution pattern was observed throughoutthe cells, including the cytoplasm and in the nucleus, in cells treatedwith the complexed GMC-5-193. DIC images revealed that the morphology ofcells treated with the GMC-5-193 complexes differed from cells treatedwith free GMC-5-193. The former showed relatively rounded cells and thelatter showed elongated and more spread cells, similar to what isobserved with untreated cells. These results indicate that aftercomplexing with the TfRscFv/LopA or TfRScFv/LipA-HoKC GMC-5-193 can bemore efficiently taken up by the cancer cells, with greater evidence ofit intended cell killing. For further investigation, the nucleus wasstained with the nucleus specific dye DAPI (blue fluorescence) and theimages analyzed. From the three-dimensional animation of thephotographed cells, it was apparent that the GMC-5-193 greenfluorescence was distributed equally throughout the cells in cellstreated with the either of the GMC-5-193 complexes, whereas the greenfluorescence was observed more in the nucleus than in the cytoplasm, incells treated with free GMC-5-193.

In Vivo Tumor Targeting by the Ligand/Liposome Complex CarryingGMC-5-193

To generate a tumor-bearing animal, MDA435/LCC6 human melanoma cells(8×10⁶) suspended in PBS were injected intravenously into the tail veinof athymic nude mice.

Using TfRscFv/LipA/GMC-5-193:

Mice carrying MDA435/LCC6 xenograft tumors, primarily in the lung, wereinjected intravenously with free GMC-5-193, non-targeted LipA/GMC-5-193(L-GMC) or TfRscFv/LipA/GMC-5-193 (scL-GMC) at 9 mg GMC-5-193/kg permouse. The molar ratio of GMC-5-193 to LipA in the complexes was 1:2.5(equivalent to 2.8:7). The ratio of single-chain antibody fragment toliposome in each complex was 1:30 (w:w). Three hours after injection,liver and lung were excised and examined under a fluorescencemicroscope. The metastases varied in size from microscopic to smallvisible metastases. FIG. 20A shows the same field photographed inbright-field and with fluorescence. It can be clearly seen that theTfRscFv/LipA-GMC-5-193 complex is able to deliver GMC-5-193 specificallyto the tumor cells in the lung. In the bright-field image of the mousetreated with free GMC-5-193, large tumors can be observed around thelung, with metastasis to the liver. However, the fluorescent image showsvery weak fluorescence in all the tissues. Untargeted complex(LipA/GMC-5-193 (L-GMC)) showed very weak signals both in lung and liverand no tumor specificity. In contrast, in the mouse injected with theTfRscFv/LipA/GMC-5-193 complex (scL-GMC), the fluorescence signal ofGMC-5-193 was much stronger in the lung metastases (distinguishable inthe bright-field image by a more dense appearance as compared to thelighter bubbly appearance of the normal lung tissue). In addition, verylow background signal was observed in the normal liver of the mousetreated with the complexed GMC-5-193, again demonstrating tumorspecificity. These results show that tumor specific uptake of GMC-5-193after systemic administration is enhanced when GMC-5-193 is incorporatedinto the TfRscFv/LipA complex.

Using TfRscFv/LipA-HoKC/GMC-5-193

Mice carrying MDA-MB-435/LCC6 xenograft tumors, primarily in the lung,were injected intravenously with free GMC-5-193 (FIG. 20B (PanelB)) orTfRscFv/LipA-HoKC/GMC-5-193 (FIG. 20B (PanelA)) at 9 mg GMC-5-193/kg permouse. The molar ratio of GMC-5-193 to LipA-HoKC in the complexes was1:2.5 (equivalent to 2.8:7). The ratio of single-chain antibody fragmentto liposome in each complex was 1:30 (w:w). Three hours after injection,liver and lung were excised and examined under a fluorescencemicroscope. The metastases varied in size from microscopic to smallvisible metastases. The same field is photographed in bright-field andwith fluorescence. It can be clearly seen that the ligand-liposomecomplex is able to deliver GMC-5-193 specifically to the tumor cells inthe lung (FIG. 20B (Left-hand panels)). In the bright-field image of themouse treated with free GMC-5-193 (FIG. 20B (right-hand panels)), largetumors can be observed around the lung, with metastasis to the liver.However, there was very weak fluorescence in all the tissues, with nofluorescence in the metastases in the liver. These results show thattumor specific uptake of GMC-5-193 after systemic administration isenhanced when GMC-5-193 is incorporated into the TfRscFv/LipA-HoKCcomplex.

Additional results showing the tumor-specific incorporation offluorescent GMC-5-193 delivered with TtRscFv/LipA-HoKC/GMC-5-193complexes is shown in FIG. 20C. The molar ratio of GMC-5-193 toLipA-HoKC in the complexes was 1:2.5 (equivalent to 2.8:7). The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). Here also the difference between the signal in the mice whichreceived intravenous administration of the TfRscFv/LipA-HoKC/GMC-5-193and those receiving intravenous free GMC-5-193 is dramatic. Large tumorsin the liver and near the spine were present in the mouse that receivedthe free GMC-5-193. However, very little fluorescence was evident inthese tumors. In contrast, the fluorescence signal was much stronger inthe lung mets and separate tumor from the mouse that received thecomplexed GMC-5-193. These above experiments clearly demonstrate theefficient tumor targeting and delivery of systemically administeredGMC-5-193 when complexed with the ligand/liposome (with and without HoKCpeptide) complex of this invention.

In Vivo Efficacy Study

C57BL/6 mice carrying B16/F10 tumors were injected with CDDP orTfRscFv/liposome/GMC-5-193 complexes (TfRscFv/LipA/GMC-5-193 orTfRscFv/LipA-HoKC/GMC-5-193) alone or in combination with CDDP. Themolar ratio of GMC-5-193 to LipA or LipA-HoKC in the complexes was 1:1.The ratio of single-chain antibody fragment to liposome in each complexwas 1:30 (w:w). FIGS. 21A-21C show the photographs of lung of each mouseafter 3 weeks of treatment. Multiple metastases of B16/F10 were observedin the lung of mice who did not undergo treatment (UT). In FIG. 21A micewere given seven intravenous injections of GMC-5-193 at 3 mgGMC-5-193/kg/injection two or three times a week. These results showthat the complexed GMC-5-193 (with and without the HoKC peptide) wasbetter than free GMC-5-193 as a single agent treatment. In FIGS. 21B and21C, the mice also received seven intravenous injections of GMC-5-193 at3 mg GMC-5-193/kg/injection two or three times a week. In the groupsthat received CDDP, the CDDP was administered twice weekly to a total ofsix injections. The initial dose of CDDP was 2.5 mg/kg, while allsubsequent doses were 2.0 mg/kg. Mice treated with the combination ofTfRscFv/Liposome/GMC-5-193 (TfRscFv/LipA/GMC-5-193) complexes plus CDDPshowed a significant decrease in the number of lung metastases (FIG.21B). In FIG. 21C, mice treated with the combination ofTfRscFv/LipA/GMC-5-193 (scL-GMC) or TfRscFv/LipA-HoKC/GMC-5-193(scL-HK-GMC) complexes plus Cisplatin also showed a significant decreasein the number of lung metastases, compared to single agent treatment(complex or CDDP).

The results of FIG. 21B correlate with those of the Western blotanalysis for cleaved caspase-3 as a marker of apoptosis (FIG. 22). Inthe serum of untreated mice or mice treated withTfRscFv/liposome/GMC-5-193 complexes (with or without HoKC) alone,cleaved caspase-3 was not detected, showing no apoptosis, thus nosignificant effect of the small molecule therapeutic. Cleaved caspase-3was detected as only a weak band in the serum of mice that received CDDPalone. Most significantly, mice treated with the combination of thecomplexed GMC-5-193 (TfRscFv/LipA/GMC-5-193 orTfRscFv/LipA-HoKC/GMC-5-193) plus CDDP showed a high level of cleavedcaspase-3, indicating enhancement of programmed cell death by thecombination therapy.

Discussion

DU145 cells. B16/F10 cells, and to a lesser extent MDA435/LCC6 cellsshowed an enhanced sensitivity to the TfRscFv/LipA/GMC-5-193 orTfRscFv/LipA-HoKC/GMC-5-193 complex in comparison to cells treated withfree GMC-5-193 (FIG. 14). Complexing GMC-5-193 also resulted in anincreased sensitization of multiple tumor cell lines (human and mouse)to various chemotherapeutic agents. Confocal images revealed thatGMC-5-193 is more efficiently taken up by MDA-MB-435 cells as thecomplexed GMC-5-193 than as free GMC-5-193 (FIGS. 19A and 19B). Muchstronger green fluorescence was observed in the cells treated with theGMC-5-193 complex, when compared with cells treated with free GMC-5-193.Interestingly, the GMC-5-193 green fluorescence was observed throughoutthe cells equally in cells treated with the GMC-5-193 complex, whereasin cells treated with free GMC-5-193, the green fluorescence wasobserved more in the nucleus than in the cytoplasm. Incorporation ofGMC-5-193 in the liganded liposome complex elevated cellular uptake andaltered the localization pattern of GMC-193 in cells.

GMC-5-193 shows anticancer effect by binding to tubulin in cytoplasm;thus the presence of the small molecule in the cytoplasm is preferablefor anticipating an anticancer effect, in addition, the drug releaseinto the cytoplasm is followed by the subsequent release into theextracellular compartment, which can cause the bystander effect. Forthis reason, enhanced anticancer activity in cells treated with thecomplexed GMC-5-193 was examined.

As results of in vitro chemosensitization of human melanoma cells MDA-MB435 to doxorubicin, a significant increase in sensitization was observedin the cells treated with the complexed GMC-5-193 compared with cellstreated with free GMC-5-193 (FIGS. 15A-15B). These sensitization resultswere dramatically different from the results of normal human lungfibroblasts IMR-90 under the same conditions, in which GMC-5-13, eitherfree or complexed had virtually no effect (FIGS. 16A-16B). Increasedfold difference between the IC₅₀ values in cancer cells were observedwhen the cells were treated with the complexed GMC-5-193 in comparisonwith the cells treated with the free GMC-5-193. These findings indicatethat TfRscFv targets and efficiently delivers the complex to the cancercells to increase the cytotoxicity, but not to normal cells,demonstrating combination therapy with complexed GMC-5-193 can increasethe therapeutic index. Similar chemosensitization of DU145 humanprostate cancer cells to (docetaxel) TAXOTERE® was observed (FIGS.17A-17B). The fold sensitization of B16/F10 metastatic mouse melanomacells to CDDP was also 2.6 fold higher when the cells were treated withthe complex (FIG. 18).

All the results from the in vitro experiments support the observationthat GMC-5-193 complexed with the targeted liposome sensitizes cancercells more effectively to the conventional chemotherapeutics than whendelivered as free GMC-5-193.

One interest in this study was in tumor-targeting delivery of GMC-5-193after systemic administration. When TfRscFv/LipA/GMC-5-193 orTfRscFv/LipA-HoKC/GMC-5-193 is administered intravenously to athymicnude mice carrying MDA-MB-435/LCC6 xenograft tumors primarily in thelung, it can be clearly seen that the ligand-liposome complex deliversGMC-5-193 specifically to the tumor cells in the lung (FIGS. 20A-20C).The green fluorescent signal of GMC-5-193 in the lung metastases wasmuch stronger in these mice than in mice treated with free GMC-5-193.Tumor-specific uptake of GMC-5-193 after systemic administration isenhanced when the small molecule is incorporated into the ligandedliposome complexes of this invention prepared by simple mixing of thecomponents. From these results, inclusion of the small molecule in theTfRScFv/LipA or TfRScFV/LipA-HoKC complexes of this invention enhanceuptake of the small molecule specifically into the tumor cells whetherthey are primary tumor or metastases.

This tumor-targeting ability was reflected in in vivo efficacy studies.B16/F10 cells showed enhanced sensitivity when treated with thecomplexed GMC-5-193, as we described above. Therefore, C57BL/6 micecarrying B16/F10 tumors were selected as animal models for efficacystudy. As shown above, mice treated with the combination ofTfRscFv/Liposome/GMC-5-193 complexes (with or without the HoKC peptide)plus CDDP showed a significant decrease of metastases in their lung andelevated levels of cleaved caspase-3 in comparison with mice treatedwith CDDP or TfRscFv/liposome/GMC-5-193 complexes alone.

Caspases involved in apoptosis are divided into two groups: theinitiator caspases, which include caspase-2, -8, -9, and -10, and theeffector caspases, which include caspase-3, -6, and -7. Activation ofeffector caspases by initiator caspases is responsible for theproteolytic cleavage of cellular substrates including actin, lamin,poly(ADP-ribose) polymerase (PARP), and inhibitors of deoxyribonuclease(such as DFF45 or ICAD). Cleavage of those substrates degrades thechromosomes into nucleosomal fragments during apoptosis. Caspase-3 hasbeen considered as most directly correlated with apoptosis because ofits location in the protease cascade pathway. Caspase-3 is synthesizedas a 32-kD precursor that is cleaved to generate the mature formcomposed of 17-kD subunits through intermediary 20-kD and 12-kD subunits34-36 Elevated levels of the cleaved caspase-3 of 17-kD is considered amarker of programmed cell deaths.

Thus, specific and efficient targeted drug delivery of the anticancersmall molecule GMC-5-193 was achieved. These results show that TfRscFvtargets the cationic liposome-GMC-5-193 complex (with or without HoKC)to tumor cells in vitro and in vivo and enhances the antitumor effect ofthe conventional chemotherapeutics in vitro and in vivo. In particular,the lowering of the effective dose of conventional chemotherapeuticagents, with a concomitant decrease in their toxic effects, wasdemonstrated.

Example 11 Preparation and Characterization of Small Molecule (YK-3-250)Comprising-Immunoliposomes by Simple Mixing

To improve the in vitro and in vivo anticancer effects of the smallmolecule YK-3-250 (a microtubule disruptive compound), a tumor-targetingliposomal complex comprising the small molecule was prepared. Inaddition to a TfRscFv/LipA complex, a cationic liposome conjugated withendosomal disrupting peptide (LipA-HoKC) can also be prepared andstudied as described herein. The endosomal disrupting peptide HoKC mayhelp the release of YK-3-250 in the cytoplasm of the cells to affecttubulin polymerization in cytoplasm. The ligand-liposome complexpreferentially targets tumor cells due to elevated levels of thecorresponding receptor on their surface. High levels of expression ofthe ligand-liposome delivered gene were evident in primary tumors andmetastasis, but not in normal tissue such as liver, lung, bone marrow,and intestinal crypts. In this Example, a liposome complex of thepresent invention, comprising the transferrin receptor single chain(TfRscFv), was used to deliver YK-3-250 to cancer cells in vitro toevaluate the in vitro bio-efficacy of the lipoplex comprising YK-3-250.

Materials and Methods

1,2-Dioeoyl-3-trimethylammonium propane (DOTAP), dioleolylphosphatidylethanolamine (DOPE), and N-maleimido-phenylbutyrate DOPE (MPB-DOPE) werepurchased from Avanti Polar Lipids (Alabaster, Ala.). TheK[K(H)KKK]₅-K(H)KKC (HoKC) (SEQ ID NO: 2) peptide was manufactured bySigma-Genosys (The Woodlands, Tex.).

Cell Lines and Culture

The human melanoma cell line MDA-MB-435 was cultured in improved MEM(IMEM) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and50 μg/mL each of penicillin, streptomycin, and neomycin. EMEM waspurchased from MediaTech (Herndon, Va.) and the other cell culture mediaand ingredients were obtained from Biofluids (Rockville, Md.).

Preparation of TfRscF/LipA/YK-3-250 Complexes

Cationic liposomal formulation LipA (DOTAP:DOPE or DDAB:DOPE at a 1:1 to1:2 molar ratio) were prepared using the ethanol injection method asdescribed herein. The concentration is 2 mM. TfRscFv/LipA/YK-3-250complexes were prepared as follows: After 10 minutes incubation,suitably at room temperature, with rotation or stirring of a mixture ofLipA and TfRscFv (ratio of TfRscFv to LipA, 1:1 to 1:40 (wt/wt), moresuitably 1:10 to 1:30 wt/wt), the YK-3-250 at the appropriateconcentration was added, mixed by inversion or stirring and incubatedfor 10 minutes, preferably at room temperature. The molar ratio ofYK-3-250 to Liposome was from 0.2:7 to 14:7, more suitably 2:7 to 8:7,most suitably 7:7. The sizes of the complexes were determined by dynamiclight scattering at 25° C. with a ZETASIZER® 3000HS system (Malvern,United Kingdom).

Preparation of TfRscFv/LipA-HoKC/YK-3-250 Complexes

Cationic liposomal formulation and LipA-MPB (DOTAP:DOPE:MPB-DOPE orDDAB:DOPE:MPB-DOPE at a 1:1:0.1 to 1:2:0.1 molar ratios) can be preparedusing the ethanol injection method. The LipA-HoKC liposome are thenprepared using the coupling reaction between the cationic liposomescarrying the maleimide group and the peptide-carrying terminal cysteinegroup as previously described herein. An aliquot of 0.1 mmol of thepeptide with a free thiol group on cysteine is added to 2 mmol ofLipA-MPB in 10 mM HEPES (pH 7.4) solution and rotated at roomtemperature for 2 hours. The resulting LipA-HoKC will have a lipidconcentration of 1.4 mM. TfRscFv/LipA-HoKC/YK-3-250 complexes areprepared as follows: After 10 minutes incubation, preferably at roomtemperature, with rotation or stirring of a mixture of LipA-HoKC andTfRscFv (ratio of TfRscFv to LipA-HoKC, 1:1 to 1:40 (wt/wt), moresuitably 1:10 to 1:30 (wt/wt)), the YK-3-250 at the appropriateconcentration is added, mixed by inversion or stirring, and incubatedfor 10 minutes at room temperature. For animal injection, dextrose orsucrose are added to each sample to a final concentration of 1% to 20%,more suitably 5-10%. The molar ratio of YK-3-250 to Liposome will befrom 0.2:7 to 14:7, more suitably 2:7 to 8:7, most suitably 7:7. Thesizes of the complexes are determined by dynamic light scattering at 25°C. with a Zetasizer 3000HS system (Malvern, United Kingdom).

In Vitro Cell Viability and Optimization of theTfRscFv/Liposome/YK-3-250 Complex

For in vitro cytotoxicity studies, 5 to 5.5×10³ cells/well in 100 μL ofthe appropriate growth medium of each cell line were plated in a 96-wellplate. After 24 hours, the cells were washed with serum-free medium,overlaid with 100 μL of TfRscFv/LipA/YK-3-250, Lip A only or freeYK-3-250 in serum-free medium in increasing concentrations, incubatedfor 4-6 hours, preferably 5 hours, and then supplemented with FBS. Thecells were then incubated for an additional 24-72 hours, preferably 48hours at 37° C. in a humidified atmosphere containing 5% CO₂. Afterward,the wells were washed with IMEM without phenol red and thecell-viability XTT-based assay was performed according to themanufacturer's protocol (Boehringer Mannheim, Indianapolis, Ind.). Inthe presence of an electron-coupling reagent, XTT, sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonate is convened intoorange formazan by dehydrogenase in the mitochondria of living cells.The formazan absorbance, which correlates to the number of living cells,was measured at 450 nm using a microplate reader (Molecular Devices,Menlo Park, Calif.). The IC₅₀ yielding 50% growth inhibition wasinterpolated from the graph of the log of drug concentration versus thefraction of surviving cells.

Results

In Vitro Optimization of the Molar Ratio of TfRscFv/LipA/YK-3-250Complex

The TfRscFv/LipA/YK-3-250 complex was prepared and the molar ratio ofthe small molecule to liposome of the TfRscFv/LipA/YK-3-250 complex wasoptimized. The cell killing effect of complexed and free YK-3-250 atdifferent ratios of YK-3-250 to LipA on MDA-MB-435 cancer cells wascompared. The ratio of TfRscFv to LipA was 1:1 to 1:40 (wt/wt), moresuitably 1:10 to 1:30 (wt/wt). 5.5×10³ cells/well were seeded in a96-well plate and treated after 24 hours with TfRscFv/LipA/YK-3-250complexes, LipA only or free YK-3-250. The XTT assays were performed 48hours after treatment to assess cytotoxicity, and the IC₅₀ values (thedrug concentration yielding 50% growth inhibition) were calculated fromthe concentration-cell viability curve. FIG. 23A shows the IC₅₀ valuesof MDA-MB-435 for each treatment. At the range of the molar ratio ofYK-3-250 to liposome of 6:7˜8:7, a decrease in the IC₅₀ values wereobserved in cells treated with the complexed YK-3-250 in comparison withcells treated with free YK-3-250, or free LipA, reducing the IC₅₀ valuesfrom >300 nM to 16 nM, down to 8 nM at the molar ratio of YK-3-250 toliposome of 7:7.

FIG. 23B shows a comparison of the effect of free YK-3-250 andTfRscFv/LipA/YK-3-250 (scL-YK-3-250) complex on MDA-MB-435 cells, usinga YK-3-205 to liposome complex (LipA) molar ratio of 7:7, (alsoequivalent to 1:1). Comparison of the level of cell killing of complexedYK-3-250 as compared to the free small molecule indicates a ˜2-foldincrease in effectiveness with the complex of this invention.

Example 12 Preparation and Characterization of Small Molecule (ImatinibMesylate (GLEEVEC®)) Comprising-Immunoliposomes by Simple Mixing

GLEEVEC® (Imatinib Mesylate; Novartis Pharmaceuticals Corp., EastHanover, N.J.), formerly known as STI-571, is an antiproliferative agent(signal transduction inhibitor), which interferes with the pathways thatsignal the growth and proliferation of tumor cells. Imatinib Mesylateselectively inhibits a group of receptor tyrosine kinases, includingBcr-Abl, c-KIT and PDGF receptors alpha and beta, leading to disruptionin cell growth and eventual cell death.

Imatinib Mesylate has been shown to be effective in inhibiting theactivity of Bcr-Abl, a tyrosine kinase protein that is dysfunctional inthis disease and signals cells to grow and divide continuously and hasbeen approved for the treatment of patients with KIT (CD117) positiveunrespectable and/or metastatic malignant Gastrointestinal StromalTumors (GIST), a relatively rare form of cancer attributable to theactivity of c-KIT tyrosine kinase.

Because protein kinases play a critical role in cellular signaltransduction cascades, and thus are directly involved in many diseasesincluding cancer, kinase inhibitors have become the focus fordevelopment of a new class of anti-cancer therapeutics. Currently morethan 30 other kinase inhibitors in clinical trials. Imatinib Mesylatecan also be utilized in the treatment of numerous other cancers,especially those that demonstrate abnormal activity of tyrosine kinasesshown to be targeted by Imatinib Mesylate, including PDGF receptorsalpha and beta. Initial results have been encouraging, however thesedrugs are not without their toxic side effects. For Imatinib Mesylatethese include hematopoietic suppression, hepatotoxicity, and renaltoxicity as well as fluid retention (swelling around the eyes or legs),diarrhea, nausea, vomiting, fatigue, muscle cramps, muscle or bone pain,abdominal pain, and rash. Reducing these side effects would lead tosignificant improvement in patient quality of life. However, whilereduction of these toxicities is important, increased concentration ofthe therapeutic agent in the tumor cells is even more significant withrespect to therapeutic benefit. The experiments described hereindemonstrate encapsulation of Imatinib Mesylate in the immunoliposomes ofthe present invention, and action in human breast, prostate andpancreatic cancer models.

Materials and Methods

1,2-Dioeoyl-3-trimethylammonium propane (DOTAP), dioleolylphosphatidylethanolamine (DOPE), and N-maleimido-phenylbutyrate DOPE (MPB-DOPE) werepurchased from Avanti Polar Lipids (Alabaster, Ala.).

Cell Lines and Culture

The human prostate cancer cell line DU145 (HTB-81) and mouse melanomacell line B16/F10 (CRL-6475) were obtained from the American TypeCulture Collection (ATCC; Manassas, Va.). DU145 was cultured in Eagleminimum essential medium with Earls salts (EMEM) supplemented with 10%heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 50μg/mL each of penicillin, streptomycin, and neomycin. B16/F10 (ATCC,CRL-6475) was cultured in Dulbecco modified Eagle medium (DMEM)supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 50μg/mL each of penicillin, streptomycin, and neomycin. The human melanomacell line MDA-MB-435 and human pancreatic cancer cell line PANC-1 werecultured in improved MEM (IMEM) supplemented with 10% heat-inactivatedFBS, 2 mM L-glutamine, and 50 μg/mL each of penicillin, streptomycin,and neomycin. Normal (non-cancerous) skin fibroblast cell line H500 wascultured in EMEM supplemented with 1 mM Sodium pryuvate, 1 mMNon-essential amino acids plus 10% heat inactivated fetal bovine serum,2 mM L-glutamine and 50 μg/mL each of penicillin, streptomycin, andneomycin. EMEM was purchased from MediaTech (Herndon, Va.) and the othercell culture media and ingredients were obtained from Biofluids(Rockville, Md.).

Preparation of TfRscFv/LipA/Imatinib Mesylate (GLEEVEC®) Complexes

Cationic liposomal formulation LipA (DOTAP:DOPE or DDAB:DOPE at a 1:1 to1:2 molar ratio) were prepared using the ethanol injection method asdescribed throughout. TfRscFv/LipA/Imatinib Mesylate complexes wereprepared as follows. After 10 minutes incubation with rotation orstirring at room temperature of a mixture of LipA and TfRscFv (ratio ofTfRscFv to LipA, 1:1 to 1:40 (wt/wt), more suitably 1:10 to 1:30 wt/wt),the Imatinib Mesylate at the appropriate concentration was added mixedby inversion or stirring at room temperature and incubated for 10minutes. For animal injection, dextrose or sucrose was added to eachsample to a final concentration of 1% to 20%, more suitably 5-10%. Themolar ratio of Imatinib Mesylate to Liposome was from 0.2:7 to 14:7,more suitably 2:7 to 8:7, most suitably 7:7. The sizes of the complexeswere determined by dynamic light scattering at 25° C. with a ZETASIZER®3000HS system (Malvern, United Kingdom).

In Vitro Cell Viability with the TfRscFv/LipA/Imatinib Mesylate Complex

For in vitro cytotoxicity studies, 2.5 to 5.5×10³ cells/well in 100 μLof the appropriate growth medium of each cell line were plated in a96-well plate. After 24 hours, the cells were washed with serum-freemedium, overlaid with 100 μL of TfRscFv/LipA/Imatinib Mesylate complexor free Imatinib Mesylate in serum-free medium in increasingconcentrations, incubated for 4-6 hours, suitably 5 hours, and thensupplemented with FBS. The cells were then incubated for an additional24-72 hours, suitably 48 hours at 37° C. in a humidified atmospherecontaining 5% CO₂. Afterward, the wells were washed with IMEM withoutphenol red and the cell-viability XTT-based assay was performedaccording to the manufacturer's protocol (Boehringer Mannheim,Indianapolis, Ind.). In the presence of an electron-coupling reagent,XTT, sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonate is converted into orange formazan by dehydrogenase in themitochondria of living cells. The formazan absorbance, which correlatesto the number of living cells, was measured at 450 nm using a microplatereader (Molecular Devices, Menlo Park, Calif.). The IC₅₀ yielding 50%growth inhibition was interpolated from the graph of the log of drugconcentration versus the fraction of surviving cells.

In Vitro Chemosensitization

For the chemosensitization study, 2.5˜5.5×10³ cells/well in 100 μL wereseeded in a 96-well plate. After 24 hours, the cells were washed withserum-free medium, overlaid with 100 μL of TfRscFv/LipA/ImatinibMesylate complex or free Imatinib Mesylate at 15 to 30 μM ImatinibMesylate, incubated for 4-6 hours, suitably 5 hours, and then FBS wasadded to each well. The cells were incubated for an additional 24-72hours, suitably 19 hours, followed by the addition of the appropriatesupplemented medium with or without chemotherapeutics in increasingconcentrations, and incubation continued for approximately 24-72 hours,suitably 48 hours. The chemotherapeutic drugs used were doxorubicin(Bedford Labs, Bedford, Ohio), docetaxel (TAXOTERE®; AventisPharmaceuticals, Bridgewater, N.J.), mitoxantrone (NOVANTRONE®, ImmunexCorp., Seattle Wash.), cisplatin (CDDP; Bedford Labs, Bedford, Ohio),gemcitabine (GEMZAR®, Eli Lilly and Co., Indianapolis Ind.) andDacarbazine (DTIC) (Mayne Pharmaceuticals, Paramus N.J.). The XTT assayswere performed to assess the degree of sensitization to thechemotherapeutics, and IC₅₀ values of each cell were calculated. Foldsensitization equals the following: IC₅₀ untransfected/IC₅₀ eachcomplex.

In Vivo Efficacy Studies

Mouse melanoma cells B16/F10 (1×10⁵) suspended in PBS were injectedintravenously into the tail vein of C57BL/6 mice. Four days later themice carrying B16/F10 tumors were intravenously injected with freeImatinib Mesylate; CDDP only or TfRscFv/LipA/Imatinib Mesylate incombination with CDDP at a dose of 0.5 mg/kg Imatinib Mesylate.Injection occurred three times a week to at total of 9 injections. Themolar ratio of Imatinib Mesylate to LipA in each complex was 7 to 7. Theratio of single-chain antibody fragment to liposome in each complex was1:30 (w:w). Certain groups also received CDDP only. CDDP was given astwice weekly injections at 2 mg/kg to a total of 6 injections. After 3weeks of treatment, the mice were sacrificed, the lungs were excised.The organs were fixed in 10% formaldehyde and preserved in 70% ethanolbefore being photographed.

Results

Comparison of Encapsulated and Free Imatinib Mesylate

The effectiveness of TfRscFv/LipA/Imatinib Mesylate (GLEEVEC®) comparedto that of free Imatinib Mesylate (GLEEVEC®), on cell survival wasassessed via XTT assay (as described herein) in both a human prostatecancer cell line (DU145) (FIG. 24A) and a human melanoma cell line(MDA-MB-435) (FIG. 24B), and a mouse melanoma cell line (B16/F10) (FIG.24C). The molar ratio of Imatinib Mesylate to LipA in each complex was 7to 7. The ratio of single-chain antibody fragment to liposome in eachcomplex was 1:30 (w:w). Imatinib Mesylate was dissolved in either DMSOor in water. As demonstrated in FIG. 24A, treatment of DU145 cell withliposome complex delivered Imatinib Mesylate resulted in a greater than5 fold increase in cell killing as compared to free Imatinib Mesylatewhen dissolved in either DMSO or water.

Similarly, in human melanoma cells, (FIG. 24B) Imatinib Mesylatedelivered via the liposome complex had a significantly greater effect oncell kill than when delivered in ‘free’ form. There was a 3 foldimprovement with TfRscFv/LipA/Imatinib Mesylate over free smallmolecule. The results were identical irrespective of whether Gleevec wasdissolved in water or DMSO. Thus subsequent experiments utilized Gleevecthat had been dissolved in water.

When the effect of free or TfRScFv/LipA complexed Imatinib Mesylate(scL-Gleevec) was compared in B16/F10 mouse melanoma cells a similar 3fold increase in cell killing with the scL-Gleevec over that with freeGleevec was observed (FIG. 24C).

Chemosensitization by TfRscFv/LipA/Imatinib Mesylate (scL-Gleevec)

The ability of Imatinib Mesylate, delivered either ‘free’ or vialiposome complex (scL-GLEEVEC®), to sensitize tumor cells (human andmouse) to first line chemotherapeutic agents was also assessed by XTTassay. Human melanoma cell line MDA-MB-435 was treated with 20 μM or 30μM free or complexed Imatinib Mesylate followed by addition ofincreasing doses of (docetaxel) TAXOTERE®. The molar ratio of ImatinibMesylate to LipA in each complex was 7 to 7. The ratio of single-chainantibody fragment to liposome in each complex was 1:30 (w:w). As shownin FIG. 25A, at a 20 uM Imatinib Mesylate concentration there is analmost 50 fold increase in response to the chemotherapeutic agent whenImatinib Mesylate is delivered by liposome complex (scL-GLEEVEC®), ascompared to administered as free Imatinib Mesylate. At this dose freeImatinib Mesylate does not sensitize the cells to (docetaxel) TAXOTERE®.Moreover a dose dependent increase in chemosensitization was observed.At 30 uM Imatinib Mesylate, while free Imatinib Mesylate showed anapproximate 4 fold sensitization, the response with liposome complexImatinib Mesylate was so dramatic that an IC₅₀ value could not bedetermined (FIG. 25B).

Similarly in human prostate cells (DU145), Imatinib Mesylate deliveredby the liposome complex (scL-GLEEVEC®) at 20 μM enhanced the response ofthe cells to Mitoxantrone by ˜4 fold (FIG. 26A) compared to freeImatinib Mesylate. Here also there was a dose response, at 30 μM an IC₅₀value could not be determined with the TfRscFv/LipA/Imatinib Mesylatecomplex (scL-GLEEVEC®) (FIG. 26B). The molar ratio of Imatinib Mesylateto LipA in each complex was 7 to 7. The ratio of single-chain antibodyfragment to liposome in each complex was 1:30 (w:w).

The effect of liposome complex delivery of Imatinib Mesylate was alsoassessed in human pancreatic cancer cell line PANC-1. The molar ratio ofImatinib Mesylate to LipA in each complex was 7 to 7. The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). As was observed in the other tumor cells, there was a doseresponse and a dramatic increase in the level of sensitivity tochemotherapeutic agent GEMZAR® (Gemcitabine HCl) when Imatinib Mesylatewas delivered via the liposome complex of the present invention(scL-GLEEVEC®) (FIGS. 27A and B). At 20 μM, there was a 100 foldincrease in the response of PANC-1 to Gemcitabine with liposome complexImatinib Mesylate over that seen with free Imatinib Mesylate (FIG. 27A).As above, at 30 μM no IC₅₀ value could be determined (FIG. 27B). Incontrast, the IC₅₀ value with the free Imatinib Mesylate did notsignificantly change with increasing dose.

The results with a mouse melanoma cell line, B16/F10, mirror those withthe human tumor cells. The comparison of the effect onchemosensitization by free and TfRscFv/LipA/Imatinib Mesylate complex(scL-GLEEVEC®) was shown to be dose and time dependent with two separatechemotherapeutic agents Cisplatin (CDDP and Dacarbazine (DTIC). Themolar ratio of Imatinib Mesylate to LipA in each complex was 7 to 7. Theratio of single-chain antibody fragment to liposome in each complex was1:30 (w:w). With CDDP, transfection with the TfRscFv/LipA/ImatinibMesylate complex (scL-GLEEVEC®) at a concentration of 20 uM resulted ina two fold increase in sensitization as compared to free ImatinibMesylate (FIG. 28A). Here again at an Imatinib Mesylate concentration of30 uM, the level of sensitization to CDDP was so strong that only ˜10%of the cells survived at the lowest Imatinib Mesylate dose (FIG. 28B).

Similar experiments were performed to compare the sensitization ofB16/F10 cells to another chemotherapeutic agent, Dacarbazine (DTIC), byfree or complexed Imatinib Mesylate. The molar ratio of ImatinibMesylate to LipA in each complex was 7 to 7. The ratio of single-chainantibody fragment to liposome in each complex was 1:30 (w:w). Here also,in addition to a dose dependent enhancement of sensitization withTfRscFv/LipA/Imatinib Mesylate, which is not seen with the free smallmolecule, there was also an increase in fold sensitization withincreasing incubation time after transfection before adding thechemotherapeutic agent (FIG. 29A to D). After 24 hours incubation, at anImatinib Mesylate concentration of 15 uM, the complexed ImatinibMesylate resulted in a 2.1 fold increase in response to DTIC as comparedto free Imatinib Mesylate (FIG. 29A). This doubled to a 4.2 foldincrease in sensitization by the complex of this invention over the freeImatinib Mesylate when the concentration of the small molecule wasincreased to 20 uM (FIG. 29B). At the longer incubation time (48 hours),the increase in sensitization with the TfRscFv/LipA/Imatinib Mesylate,as compared to free Imatinib Mesylate (concentration=15 uM) was 5.7 fold(FIG. 29C), while at 48 hours with a concentration of 20 uM the responsewas so great no IC₅₀ is obtainable (FIG. 29D). Thus, here again we haveshown in multiple tumor cell types and with various chemotherapeuticagents the delivery and efficacy of the small molecule Imatinib Mesylateis enhanced when it is encapsulated in the complex of this invention.Imatinib Mesylate.

The tumor cell specific nature of the ligand/liposome/Imatinib Mesylateinduced chemosensitization is shown in FIGS. 30 A and B. Normal(non-cancerous) skin fibroblast cells (H500) were transfected as abovewith LipA only, free Imatinib Mesylate and TfRscFv/LipA/ImatinibMesylate (scL-GLEEVEC®) (at a concentration of 20 uM) prior to theaddition of chemotherapeutic agents Mitoxantrone (FIG. 30A) or(docetaxel) TAXOTERE® (FIG. 30B). The molar ratio of Imatinib Mesylateto LipA in each complex was 7 to 7. The ratio of single-chain antibodyfragment to liposome in each complex was 1:30 (w:w). In both experimentsno significant increase in sensitization to the chemotherapeutic agentswas observed. All IC₅₀ values were in the same range as that of thecontrols, >100 ng/ml for mitoxantrone and >100 nM for (docetaxel)TAXOTERE®.

In Vivo Efficacy of the TfRscFv/LipA/Imatinib Mesylate Complex: EnhancedTumor Growth Inhibition

1×10⁵ B16/F10 mouse melanoma cells, suspended in PBS were injectedintravenously into the tail vein of C57BL/6 mice. Four days later, themice carrying B16/F10 lung metastases were injected with eithercisplatin (CDDP) only, free Imatinib Mesylate or TfRscFv/LipA/ImatinibMesylate (scL-GLEEVEC®) complex in combination with cisplatin. The molarratio of Imatinib Mesylate to LipA in each complex was 7 to 7. The ratioof single-chain antibody fragment to liposome in each complex was 1:30(w:w). The mice received three injections a week of free ImatinibMesylate or scL-GLEEVEC® complex at 0.5 mg ImatinibMesylate/mouse/injection to a total of 9 injections. CDDP was i.p. twicea week at a dose of 2 mg/kg to a total of 6 injections. After 3 weeks oftreatment, the mice were humanely euthanized and the lungs were excisedand photographed.

As shown in FIG. 31, there was a significant inhibition of tumor cellgrowth in the lungs of the mice that received the combination ofTfRScFv/Lipa/Imatinib Mesylate plus CDDP compared to those from theanimals that received single agent treatment. Thus, this supports theuse of the ligand/liposome/small molecule complex of this invention asan anticancer agent, particularly when used in combination withchemotherapeutic agents.

Conclusions

Imatinib Mesylate has been approved for treating patients withPhiladelphia chromosome-positive (Ph+) Chronic Myeloid Leukemia (CML)and for patients with KIT (CD117) positive unrespectable and/ormetastatic malignant Gastrointestinal Stromal Tumors (GIST). It has beenshown in vitro to inhibit a number of tyrosine kinases, includingBcr-Abl, c-KIT and PDGF receptors alpha and beta. Thus, the drug haspotential for the treatment of other cancers that express these kinases.In these studies, we have demonstrated that the effectiveness ofImatinib Mesylate in inducing tumor cell death (in breast, prostate andpancreatic cancers) and increasing their response to first linechemotherapeutic agents, is dramatically improved when delivered to thecells via the targeted liposome delivery complexes of the presentinvention. These results also demonstrate the ability of thisnanocomplex to greatly increase the efficacy of Imatinib Mesylate.

Example 13 Preparation and Characterization of Small Molecule (Erlotinib(TARCEVA®)) Comprising-Immunoliposomes by Simple Mixing

TARCEVA® (Erlotinib, Genentech Inc, So. San Francisco Calif.) is a HumanEpidermal Growth Factor Receptor Type 1/Epidermal Growth Factor Receptor(HER1/EGFR) tyrosine kinase inhibitor. This tyrosine kinase is one ofthe factors critical to cell growth in non-small cell lung andpancreatic cancers.

The epidermal growth factor receptor (EGFR) is a component of the HER(human epidermal growth factor receptor) signaling pathway. The pathwayconsists of at least four cellular receptors: EGFR/HER1, HER2, HER3, andHER4. Approximately 11 different factors are known to bind and activatethese receptors in certain patterns. The HER signaling pathway plays arole in the normal regulation of cell growth, proliferation, migration,and mediating processes, such as wound healing, tissue repair, andmaintenance of the skin. In addition to its role in controlling thegrowth of normal cells, the HER signaling pathway has been shown to havea significant impact on the growth, proliferation, migration, andsurvival of cancer cells.

EGFR and other components of the HER signaling pathway interact in acomplex and tightly regulated, manner to regulate cell growth.Alterations in the amount or activity of HER family members may cause orsupport the inappropriate cell growth that leads to proliferation,migration, and survival of cancer cells. Because the signaling pathwayworks as a cascade that amplifies the growth signal at each step, smallchanges in the amount or activity of EGFR may significantly drive thedevelopment, or progression, of cancer by promoting cell growth andmetastasis (cell migration) and inhibiting apoptosis (programmed celldeath). Additionally, several studies have shown that HER signaling, animportant regulator of normal cellular and tissue repair, is activatedin response to a variety of cancer therapies that damage cells andtissues, including some chemotherapeutic agents and radiation. Thesestudies suggest that activation of the HER pathway, including EGFR, maycontribute to the development of treatment-resistant cancers.

TARCEVA® is designed to inhibit the tyrosine kinase activity of the HER1signaling pathway inside the cell. TARCEVA® is an oral dosage tablet.Erlotinib is a quinazolinamine with the chemical nameN-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine. TARCEVA®contains erlotinib as the hydrochloride salt. Erlotinib inhibits theintracellular phosphorylation of tyrosine kinase associated with theepidermal growth factor receptor (EGFR). Specificity of inhibition withregard to other tyrosine kinase receptors has not been fullycharacterized. EGFR is expressed on the cell surface of normal cells andcancer cells. In November 2004, the U.S. Food and Drug Administration(FDA) approved TARCEVA® (150 mg) for the treatment of patients withlocally advanced or metastatic non-small cell lung cancer (NSCLC) afterfailure of at least one prior chemotherapy regimen. Results from twomulticenter, placebo-controlled, randomized Phase ill trials conductedin first-line patients with locally advanced or metastatic NSCLC showedno clinical benefit with the concurrent administration of TARCEVA® withplatinum-based chemotherapy, and its use is not recommended in thatsetting. In this regard, the increase in sensitization to variouschemotherapeutic agents in multiple tumor cell lines by Erlotinib(TARCEVA®) through the method of this invention, i.e. complexing thesmall molecule with the tumor targeting liganded liposome complexthrough simple mixing, is highly unexpected. The most common sideeffects in patients with NSCLC receiving TARCEVA® monotherapy 150 mgwere mild-to-moderate rash and diarrhea. Grade 3/4 rash and diarrheaoccurred in 9 and 6 percent of TARCEVA®-treated patients, respectively,with each resulting in 1 percent of patients discontinuing thesingle-agent Phase III trial. There have been infrequent reports ofserious Interstitial Lung Disease (ILD)-like events, includingfatalities, in patients receiving TARCEVA® for treatment of NSCLC,pancreatic cancer or other advanced solid tumors. In the NSCLCsingle-agent trial, the incidence of ILD-like events were infrequent(0.8 percent) and were equally distributed between treatment arms.

In November 2005, the FDA approved TARCEVA® (100 mg) in combination withgemcitabine chemotherapy for the treatment of locally advanced,inoperable or metastatic pancreatic cancer in patients who have notreceived previous chemotherapy. In the Phase III study in pancreaticcancer, the most common adverse events reported were fatigue, rash,nausea, anorexia and diarrhea. Rash was reported in 69 percent ofpatients who received TARCEVA® plus gemcitabine and in 30 percent ofpatients who received gencitabine plus placebo. Diarrhea was reported in48 percent of patients who received Tarceva plus gemcitabine and in 36percent of patients who received gemcitabine plus placebo. Rash anddiarrhea each resulted in dose reductions in two percent of patients,and resulted in study discontinuation in up to one percent of patientswho received TARCEVA® plus gemcitabine. In addition, severe andpotential fatal adverse events included interstitial lung disease-likecomplications, myocardial infarction or ischemia, cerebrovascularaccident, and microangiopathic hemolytic anemia with thrombocytopenia ofpatients who received TARCEVA® plus gemcitabine.

Materials and Methods

1,2-Dioeoyl-3-trimethylammonium propane (DOTAP), dioleolylphosphatidylethanolamine (DOPE), and N-maleimido-phenylbutyrate DOPE (MPB-DOPE) werepurchased from Avanti Polar Lipids (Alabaster, Ala.).

Cell Lines and Culture

The human prostate cancer cell line DU145 (HTB-81) was obtained from theAmerican Type Culture Collection (ATCC, Manassas, Va.). DU145 wascultured in Eagle minimum essential medium with Earls salts (EMEM)supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mML-glutamine, and 50 μg/mL each of penicillin, streptomycin, andneomycin. The human melanoma cell line MDA-MB-435 and human pancreaticcancer cell line PANC-1 were cultured in improved MEM (IMEM)supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 50μg/mL each of penicillin, streptomycin, and neomycin. Normal(non-cancerous) skin fibroblast cell line H500 was cultured in EMEMsupplemented with 1 mM Sodium pryuvate, 1 mM Non-essential amino acidsplus 10% heat inactivated fetal bovine serum, 2 mM L-glutamine and 50μg/mL each of penicillin, streptomycin, and neomycin. EMEM was purchasedfrom MediaTech (Herndon, Va.) and the other cell culture media andingredients were obtained from Biofluids (Rockville, Md.).

Preparation of TfRscFv/LipA/Erlotinib (TARCEVA®) Complexes

Cationic liposomal formulation LipA (DOTAP:DOPE or DDAB:DOPE at a 1:1 to1:2 molar ratio) were prepared using the ethanol injection method asdescribed throughout. TfRscFv/LipA/Erlotinib complexes were prepared asfollows: After 10 minutes incubation with rotation or stirring at roomtemperature of a mixture of LipA and TfRscPv (ratio of TfRscFv to LipA,1:1 to 1:40 (wt/wt), more suitably 1:10 to 1:30 wt/wt), the Erlotinib atthe appropriate concentration was added mixed by inversion or stirringat room temperature and incubated for 10 minutes. For animal injection,dextrose or sucrose was added to each sample to a final concentration of1% to 20%, more suitably 5-10%. The molar ratio of Erlotinib to Liposomewas from 0.2:7 to 14:7, more suitably 2:7 to 8:7, most suitably 7:7. Thesizes of the complexes were determined by dynamic light scattering at25° C. with a ZETASIZER® 3000HS system (Malvern, United Kingdom).

In Vitro Cell Viability with the TfRscFv/LipA/Erlotinib Complex

For in vitro cytotoxicity studies, 2.5 to 3.5×10³ cells/well in 100 μLof the appropriate growth medium of each cell line were plated in a96-well plate. After 24 hours, the cells were washed with serum-freemedium, overlaid with 100 μL of TfRscFv/LipA/Erlotinib complex or freeErlotinib in serum-free medium in increasing concentrations, incubatedfor 4-6 hours, suitably 5 hours, and then supplemented with FBS. Thecells were then incubated for an additional 24-72 hours, suitably 48hours at 37° C. in a humidified atmosphere containing 5% CO₂. Afterward,the wells were washed with IMEM without phenol red and thecell-viability XTT-based assay was performed according to themanufacturer's protocol (Boehringer Mannheim, Indianapolis, Ind.). Inthe presence of an electron-coupling reagent, XTT, sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonate is converted intoorange formazan by dehydrogenase in the mitochondria of living cells.The formazan absorbance, which correlates to the number of living cells,was measured at 450 nm using a microplate reader (Molecular Devices,Menlo Park, Calif.). The IC₅₀ yielding 50% growth inhibition wasinterpolated from the graph of the log of drug concentration versus thefraction of surviving cells.

In Vitro Chemosensitization

For the chemosensitization study, 2.5˜3.5×10³ cells/well in 100 μL wereseeded in a 96-well plate. After 24 hours, the cells were washed withserum-free medium, overlaid with 100 μL of TfRscFv/LipA/Erlotinibcomplex or free Erlotinib at 3 to 8 PM Erlotinib, incubated for 4-6hours, suitably 5 hours, and then FBS was added to each well. The cellswere incubated for an additional 24-72 hours, suitably 19 hours,followed by the addition of the appropriate supplemented medium with orwithout chemotherapeutics in increasing concentrations, and incubationcontinued for approximately 24-72 hours, suitably 48 hours. Thechemotherapeutic drugs used were docetaxel (TAXOTERE®; AventisPharmaceuticals, Bridgewater, N.J.), mitoxantrone (NOVANTRONE®, ImmunexCorp., Seattle Wash.) and gemcitabine (GEMZAR®, Eli Lilly and Co.,Indianapolis Ind.). The XTT assays were performed to assess the degreeof sensitization to the chemotherapeutics, and IC₅₀ values of each cellwere calculated. Fold sensitization equals the following: IC₅₀untransfected/IC₅₀ each complex.

Results

Comparison of Encapsulated and Free Erlotinib (TARCEVA®)

The effectiveness of TfRscFv/LipA/Erlotinib (TARCEVA®) compared to thatof free Erlotinib (TARCEVA®) on cell survival was assessed via XTT assay(as described herein) in a human prostate cancer cell line (DU14S) (FIG.32A), a human pancreatic cancer cell line (PANC-1) (FIG. 32B) and ahuman melanoma cell line (MDA-MB-435) (FIG. 32C). The molar ratio ofErlotinib (TARCEVA®) to LipA in each complex was 7 to 7. The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). Erlotinib (TARCEVA®) was dissolved in water. As demonstrated inFIG. 32A, treatment of DU145 cell with liposome complex deliveredErlotinib (TARCEVA®) resulted in a greater than 5 fold increase in cellkilling as compared to free when Erlotinib (TARCEVA®).

Similarly, in human pancreatic cancer cells, (FIG. 32B) Erlotinib(TARCEVA®) delivered via the liposome complex had a significantlygreater effect on cell kill than when delivered in ‘free’ form. Therewas an almost 10 fold improvement with TfRscFv/LipA/Erlotinib (TARCEVA®)over free small molecule.

When the effect of free or TfRScFv/LipA complexed Erlotinib (TARCEVA®)(scL-Tarceva) was compared in human melanoma cells (MDA-MB-435) a >2fold increase in cell killing with the scL-TARCEVA® over that with freeTarceva was observed (FIG. 32C).

Chemosensitization by TfRscFv/LipA/Erlotinib (scL-TARCEVA®)

The ability of Erlotinib (TARCEVA®), delivered either ‘free’ or vialiposome complex (scL-TARCEVA®), to sensitize tumor cells to first linechemotherapeutic agents was also assessed by XTT assay. Human prostatecell line DU145 was treated with 3.75 μM or 7.5 μM free or complexedErlotinib (TARCEVA®) followed by addition of increasing doses ofMitoxantrone. The molar ratio of Erlotinib (TARCEVA®) to LipA in eachcomplex was 7 to 7 (equivalent to 1:1). The ratio of single-chainantibody fragment to liposome in each complex was 1:30 (w:w). As shownin FIG. 33A, at a 3.75 uM Erlotinib (TARCEVA®) concentration there is analmost 13 fold increase in response to the chemotherapeutic agent whenErlotinib (TARCEVA®) is delivered by liposome complex (scL-TARCEVA®), ascompared to administered as free Erlotinib. At this dose free Erlotinibdoes not sensitize the cells to Mitoxantrone. Moreover a dose dependentincrease in chemosensitization was observed, but only when Erlotinib(TARCEVA®) was delivered via the method of this invention and not asfree Erlotinib (TARCEVA®). At 7.5 uM Erlotinib (TARCEVA®), the level ofsensitization of DU45S cells to Mitoxantrone after treatment withscL-TARCEVA® was over 34 fold higher than that of free Erlotinib(TARCEVA®) (FIG. 33B). In contrast, even at this higher dose freeErlotinib (TARCEVA®) had no effect on the response to thechemotherapeutic agent.

Similarly in human melanoma cells (MDA-MB-435), Erlotinib (TARCEVA®)delivered by the liposome complex (scL-TARCEVA®) at 3.75 μM enhanced theresponse of the cells to (docetaxel) TAXOTERE® by >4 fold (FIG. 34A)compared to free Erlotinib (TARCEVA®). Here also there was a doseresponse, but once again only with the scL delivered small molecule. At7.5 μM the level of sensitization was so strong that an IC₅₀ value couldnot be determined with the TFRscFv/LipA/Erlotinib (TARCEVA®) complex(scL-TARCEVA®) (FIG. 34B), but the fold sensitization as compared tofree Erlotinib is estimated to be >140 fold. In contrast there was onlyminimal effect of free Erlotinib. The molar ratio of Erlotinib(TARCEVA®) to LipA in each complex was 7 to 7. The ratio of single-chainantibody fragment to liposome in each complex was 1:30 (w:w).

The effect of liposome complex delivery of Erlotinib (TARCEVA®) was alsoassessed in human pancreatic cancer cell line PANC-1. The molar ratio ofImatinib Mesylate to LipA in each complex was 7 to 7. The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). As was observed in the other tumor cells, there was a dramaticincrease in the level of sensitivity to chemotherapeutic agent GEMZAR®(Gemcitabine HCl) when Erlotinib (TARCEVA®) was delivered via theliposome complex of the present invention (scL-TARCEVA®) (FIG. 35). At7.5 M, there was an amazing 545 fold increase in the response of PANC-1to Gemcitabine with liposome complexed Erlotinib (TARCEVA®) over thatseen with free Erlotinib. In light of what has been found in clinicaltrials where free TARCEVA® was found not to enhance the effect ofstandard chemotherapeutics, such a dramatic effect on response tostandard chemotherapeutics is highly unexpected.

Thus, here again as with the other small molecules in the previousexamples, we have shown in multiple tumor cell types and with variouschemotherapeutic agents that the delivery and efficacy of the smallmolecule Erlotinib is enhanced when it is encapsulated in the complex ofthis invention.

The tumor cell specific nature of the ligand/liposome/Erlotinib(TARCEVA®) induced chemosensitization is shown in FIGS. 36 A-C. Normal(non-cancerous) skin fibroblast cells (H500) were transfected as abovewith LipA only, free Erlotinib (TARCEVA® alone) andTfRscFv/LipA/Erlotinib (TARCEVA®) (scL-TARCEVA®) (at a concentration of7.5 uM) prior to the addition of chemotherapeutic agents Mitoxantrone(FIG. 36A), (docetaxel) TAXOTERE® (FIG. 36B) or Gemcitabine (GEMZAR®)(FIG. 36C). The molar ratio of Erlotinib (TARCEVA®) to LipA in eachcomplex was 7 to 7. The ratio of single-chain antibody fragment toliposome in each complex was 1:30 (w:w). In all 3 experiments nosignificant increase in sensitization to the chemotherapeutic agentswere observed. All IC₅₀ values were in the same range as that of thecontrols.

Thus, here again as with the other small molecules in the previousexamples, we have shown in multiple tumor cell types and with variouschemotherapeutic agents that the delivery and efficacy of the smallmolecule Erlotinib is enhanced when it is encapsulated in the complex ofthis invention.

Example 14 Preparation and Characterization of Small Molecule SunitinibMalate (SUTENT®) Comprising-Immunoliposomes by Simple Mixing

The majority of cancers, including renal cell carcinoma (RCC) andgastrointestional stromal tumor (GIST), result from mutations or otherabnormalities in multiple signaling pathways, as opposed to a single,well defined mutation or abnormality. These changes ultimately enablethe processes critical to cancer growth and include: self-sufficiency ingrowth signals, insensitivity to growth-inhibitory signals, evasion ofprogrammed cell death (apoptosis), limitless replicative potential,sustained angiogenesis, and tissue invasion and metastasis. Sunitinibmalate (SUTENT®) is an oral multi-kinase inhibitor that simultaneouslyinhibits several receptor kinases involved in RCC and GIST development.Sunitinib malate (SUTENT®) simultaneously inhibits all known PDGF andVEGF receptors, which play a role in both tumor cell proliferation andangiogenesis. SUTENT® is the first multi-kinase GIST therapy tosimultaneously inhibit PDGF, VEGF, and KIT receptors. SUTENT®demonstrated antiangiogenic activity by inhibiting PDGF receptors onpericytes and VEGP receptors on endothelial cells in preclinical studiesin both RCC and GIST. SUTENT® induced tumor regression and inhibitedangiogenesis and metastatic progression in preclinical studies. SUTENT®,therefore inhibits multiple signaling pathways, resulting in adual-action antiproliferative and antiangiogenic effect.

SUTENT® is indicated for the treatment of gastrointestinal stromal tumor(GIST) after disease progression on or intolerance to imatinib mesylate,and for the treatment of advanced renal cell carcinoma (RCC). Approvalfor advanced RCC is based on partial response rates and duration ofresponses. There are no randomized trials of SUTENT® demonstratingclinical benefit such as increased survival or improvement indisease-related symptoms in RCC.

Materials and Methods

1,2-Dioeoyl-3-trimethylammonium propane (DOTAP), dioleolylphosphatidylethanolamine (DOPE), and N-maleimido-phenylbutyrate DOPE (MPB-DOPE) werepurchased from Avanti Polar Lipids (Alabaster, Ala.).

Cell Lines and Culture

The human prostate cancer cell line DU145 (HTB-81) was obtained from theAmerican Type Culture Collection (ATCC; Manassas, Va.). DU145 wascultured in Eagle minimum essential medium with Earls salts (EMEM)supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mML-glutamine, and 50 μg/mL each of penicillin, streptomycin, andneomycin. The human melanoma cell line MDA-MB-435 and human pancreaticcancer cell line PANC-1 were cultured in improved MEM (IMEM)supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 50μg/mL each of penicillin, streptomycin, and neomycin. Normal human lungfibroblast IMR-90 cells, a gift from Dr. I. Panyutin (Nuclear MedicineDepartment, National Institutes of Health, Bethesda, Md.), were culturedin EMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine,0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 50 μg/mL eachof penicillin, streptomycin, and neomycin. Normal (non-cancerous) skinfibroblast cell line H500 was cultured in EMEM supplemented with 1 mMSodium pryuvate, 1 mM Non-essential amino acids plus 10% heatinactivated fetal bovine serum, 2 mM L-glutamine and 50 μg/mL each ofpenicillin, streptomycin, and neomycin. EMEM was purchased fromMediaTech (Herndon, Va.) and the other cell culture media andingredients were obtained from Biofluids (Rockville, Md.).

Preparation of TfRscFv/LipA/Sunitinib Malate (SUTENT®) Complexes

Cationic liposomal formulation LipA (DOTAP:DOPE or DDAB:DOPE at a 1:1 to1:2 molar ratio) were prepared using the ethanol injection method asdescribed throughout. TfRscFv/LipA/Sunitinib malate (SUTENT®) complexeswere prepared as follows: After 10 minutes incubation with rotation orstirring at room temperature of a mixture of LipA and TfRscFv (ratio ofTfRscFv to LipA, 1:1 to 1:40 (wt/wt), more suitably 1:10 to 1:30 wt/wt),the Sunitinib malate (SUTENT®) at the appropriate concentration wasadded mixed by inversion or stirring at room temperature and incubatedfor 10 minutes. For animal injection, dextrose or sucrose was added toeach sample to a final concentration of 1% to 20%, more suitably 5-10%.The molar ratio of Sunitinib malate (SUTENT®) to Liposome was from 0.2:7to 14:7, more suitably 3:7 to 11:7, most suitably 7:7. The sizes of thecomplexes were determined by dynamic light scattering at 25° C. with aZETASIZER® 3000HS system (Malvern, United Kingdom).

In Vitro Cell Viability with the TfRscFv/LipA/Sunitinib Malate (SUTENT®)Complex

For in vitro cytotoxicity studies, 2.5 to 3.5×10³ cells/well in 100 μLof the appropriate growth medium of each cell line were plated in a96-well plate. After 24 hours, the cells were washed with serum-freemedium, overlaid with 100 μL of TfRscFv/LipA/Sunitinib malate (SUTENT®)complex or free Sunitinib malate (SUTENT®) in serum-free medium inincreasing concentrations, incubated for 4-6 hours, suitably 5 hours,and then supplemented with FBS. The cells were then incubated for anadditional 24-72 hours, suitably 48 hours at 37° C. in a humidifiedatmosphere containing 5% CO₂. Afterward, the wells were washed with IMEMwithout phenol red and the cell-viability XTT-based assay was performedaccording to the manufacturer's protocol (Boehringer Mannheim,Indianapolis, Ind.). In the presence of an electron-coupling reagent,XTT, sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonate is converted into orange formazan by dehydrogenase in themitochondria of living cells. The formazan absorbance, which correlatesto the number of living cells, was measured at 450 nm using a microplatereader (Molecular Devices, Menlo Park, Calif.). The IC₅₀ yielding 50%growth inhibition was interpolated from the graph of the log of drugconcentration versus the fraction of surviving cells.

In Vitro Chemosensitization

For the chemosensitization study, 2.5˜3.5×10³ cells/well in 100 μL wereseeded in a 96-well plate. After 24 hours, the cells were washed withserum-flee medium, overlaid with 100 μL of TtRscFv/LipA/Sunitinib malate(SUTENT®) complex or free Sunitinib malate (SUTENT®) at 2 to 6 μMSunitinib malate (SUTENT®), incubated for 4-6 hours, suitably 5 hours,and then FBS was added to each well. The cells were incubated for anadditional 24-72 hours, suitably 19 hours, followed by the addition ofthe appropriate supplemented medium with or without chemotherapeutics inincreasing concentrations, and incubation continued for approximately24-72 hours, suitably 48 hours. The chemotherapeutic drugs used weredocetaxel (TAXOTERE®; Aventis Pharmaceuticals, Bridgewater, N.J.),mitoxantrone (NOVANTRONE®, Immunex Corp., Seattle Wash.) and gemcitabine(GEMZAR®, Eli Lilly and Co., Indianapolis Ind.). The XTT assays wereperformed to assess the degree of sensitization to thechemotherapeutics, and IC₅₀ values of each cell were calculated. Foldsensitization equals the following: IC₅₀ untransfected/IC₅₀ eachcomplex.

Results

In Vitro Optimization of the Molar Ratio of TfRscFv/LipA/SunitinibMalate (SUTENT®) Complex

The TfRscFv/LipA/Sunitinib malate (SUTENT®) complex was prepared and themolar ratio of the small molecule to liposome of theTfRscFv/LipA/Sunitinib malate (SUTENT®) complex was optimized. The cellkilling effect of complexed and free Sunitinib malate (SUTENT®) atdifferent ratios of Sunitinib malate (SUTENT®) to LipA on DU1455 humanprostate cancer cells was compared. The ratio of TfRscFv to LipA was1:30 (wt/wt). 3×10³ cells/well were seeded in a 96-well plate andtreated after 24 hours with TfRscFv/LipA/Sunitinib malate (SUTENT®)complexes, or free Sunitinib malate (SUTENT®). The XTT assays wereperformed 72 hours after treatment to assess cytotoxicity, and the IC₅₀values (the drug concentration yielding 50% growth inhibition) werecalculated from the concentration-cell viability curve. FIG. 37 showsthe IC₅₀ values for each ratio. At the range of the molar ratio ofSunitinib malate (SUTENT®) to liposome of 3:7˜7:7, a decrease in theIC₅₀ values were observed in cells treated with the complexed Sunitinibmalate (SUTENT®) in comparison with cells treated with free Sunitinibmalate (SUTENT®).

Comparison of Encapsulated and Free Sunitinib malate (SUTENT®)

The effectiveness of TfRscFv/LipA/Sunitinib malate (SUTENT®) compared tothat of free Sunitinib malate (SUTENT®) on cell survival was assessedvia XTT assay (as described herein) in a human prostate cancer cell line(DU145) (FIG. 38A), and a human pancreatic cancer cell line (PANC-1)(FIG. 38B). The molar ratio of Sunitinib malate (SUTENT®) to LipA ineach complex was 7 to 7 (equivalent to 1:1). The ratio of single-chainantibody fragment to liposome in each complex was 1:30 (w:w). Asdemonstrated in FIG. 38A, treatment of DU145 cell with liposome complexdelivered Sunitinib malate (SUTENT®) resulted in a significant increasein cell killing as compared to free Sunitinib malate (SUTENT®).

Similarly, in human pancreatic cancer cells, (FIG. 38B) Sunitinib malate(SUTENT®) delivered via the liposome complex had a significantly greatereffect on cell kill than when delivered in ‘free’ form. There was a 4fold improvement with TfRscFv/LipA/Sunitinib malate (SUTENT®) over freesmall molecule.

Chemosensitization by TfRscFv/LipA/Sunitinib malate (scL-SUTENT®)

The ability of Sunitinib malate (SUTENT®), delivered either ‘free’ orvia liposome complex (scL-SUTENT®), to sensitize tumor cells to firstline chemotherapeutic agents was also assessed by XTT assay. Humanmelanoma cell line MDA-MB-435 was treated with 2.5 μM or 5 μM free orcomplexed Sunitinib malate (SUTENT®) followed by addition of increasingdoses of (docetaxel) TAXOTERE®. The molar ratio of Sunitinib malate(SUTENT®) to LipA in each complex was 7 to 7 (equivalent to 1:1). Theratio of single-chain antibody fragment to liposome in each complex was1:30 (w:w). As shown in FIG. 39A, at a 2.5 uM Sunitinib malate (SUTENT®)concentration there is greater than 8 fold increase in response to thechemotherapeutic agent when delivered by Sunitinib malate (SUTENT®)liposome complex (scL-SUTENT®), as compared to administered as freeSunitinib malate (SUTENT®). Moreover a dose dependent increase inchemosensitization was observed when Sunitinib malate (SUTENT®) wasdelivered via the method of this invention. At 5 uM Sunitinib malate(SUTENT®) the level of sensitization was so high that an IC₅₀ valuecould not be reached however it was estimated that the level ofsensitization of MDA-MB-435 cells to (docetaxel) TAXOTERE® aftertreatment with scL-SUTENT® was over 300 fold higher than that of freeSunitinib malate (SUTENT®) (FIG. 39B).

Similarly in human prostate cancer cells (DU145), Sunitinib malate(SUTENT®) delivered by the liposome complex (scL-SUTENT®) at 5 μMenhanced the response of the cells to Mitoxantrone by >400 fold (FIG.40) compared to free Sunitinib malate (SUTENT®) where there was onlyminimal effect on sensitization of the prostate cancer cells to thechemotherapeutic agent. The molar ratio of Sunitinib malate (SUTENT®) toLipA in each complex was 7 to 7. The ratio of single-chain antibodyfragment to liposome in each complex was 1:30 (w:w).

The effect of liposome complex delivery of Sunitinib malate (SUTENT®)was also assessed in human pancreatic cancer cell line PANC-1. The molarratio of Sunitinib malate (SUTENT®) to LipA in each complex was 7 to 7.The ratio of single-chain antibody fragment to liposome in each complexwas 1:30 (w:w). As was observed in the other tumor cells, there was adramatic increase in the level of sensitivity to chemotherapeutic agentGEMZAR® (Gemcitabine HCl) when Sunitinib malate (SUTENT®) was deliveredvia the liposome complex of the present invention (scL-SUTENT®) (FIG.41). At 2.5 μM, there was an almost 6 fold increase in the response ofPANC-1 to Gemcitabine (GEMZAR®) with liposome complexed Sunitinib malate(SUTENT®) over that seen with free Sunitinib malate (SUTENT®).

The tumor cell specific nature of the ligand/liposome/Sunitinib malate(SUTENT®) induced chemosensitization is shown in FIGS. 42 (A-C) and 43(A-C) Normal (non-cancerous) skin fibroblast cells (H500) weretransfected as above with LipA only, free Sunitinib malate (SUTENT®)alone and TfRscFv/LipA/Sunitinib malate (SUTENT®) (scL-SUTENT®) (at aconcentration of 2.5 or 5 uM) prior to the addition of chemotherapeuticagents Mitoxantrone (FIG. 42A), (docetaxel) TAXOTERE® (FIG. 42B) orGemcitabine (GEMZAR®) (FIG. 42C). The molar ratio of Sunitinib malate(SUTENT®) to LipA in each complex was 7 to 7. The ratio of single-chainantibody fragment to liposome in each complex was 1:30 (w:w). In all 3experiments no significant increase in sensitization to thechemotherapeutic agents were observed. All ICs values were in the samerange as that of the controls.

Similar results were observed when normal human lung fibroblasts(IMR-90) cells were transfected as above with LipA only, free Sunitinibmalate (SUTENT®) alone and TfRscFv/LipA/Sunitinib malate (SUTENT®)(scL-SUTENT®) (at a concentration of 2.5 uM) prior to the addition ofchemotherapeutic agents Mitoxantrone (FIG. 43A), (docetaxel) TAXOTERE®(FIG. 43B) or Gemcitabine (GEMZAR®) (FIG. 43C). The molar ratio ofSunitinib malate (SUTENT®) to LipA in each complex was 7 to 7. The ratioof single-chain antibody fragment to liposome in each complex was 1:30(w:w). In all 3 experiments no significant increase in sensitization tothe chemotherapeutic agents were observed. All IC₅₀ values were in thesame range as that of the controls.

Thus, here again as with the other small molecules in the previousexamples, we have shown in multiple tumor cell types and with variouschemotherapeutic agents that the delivery and efficacy of the smallmolecule Sunitinib malate (SUTENT®) is enhanced when it is encapsulatedin the complex of this invention. Since the three tumor types shown inthis example to be significantly more sensitized to Sunitinib malate(SUTENT®) when complexed as described in this invention, as compared tofree Sunitinib malate (SUTENT®) are not tumor types in which the use ofthis small molecule has been found to be useful, it is an unexpectedfinding that these tumor cells respond so well to this invention.

Example 15 Preparation and Characterization of Small Molecule (Geftinib(IRESSA®)) Comprising-Immunoliposomes by Simple Mixing

Geftinib (IRESSA®) was the first in a new class of anti-cancer drugs,known as epidermal growth factor receptor tyrosine kinase (EGFR-TK)inhibitors, to gain market approval and is currently licensed for thetreatment of advanced non-small-cell lung cancer (NSCLC) in 36 countriesworldwide. Many cells, including cancer cells, have receptors on theirsurfaces for epidermal growth factor (EGF), a protein that is normallyproduced by the body and that promotes the growth and multiplication ofcells. When EGF attaches to epidermal growth factor receptors (EGFRs),it causes an enzyme called tyrosine kinase to become active within thecells. Tyrosine kinase triggers chemical processes that cause the cells,including cancer cells, to grow, multiply, and spread. Gefitinibattaches to EGFRs and thereby blocks the attachment of EGF and theactivation of tyrosine kinase. This mechanism for stopping cancer cellsfrom growing and multiplying is very different from the mechanisms ofchemotherapy and hormonal therapy. Gefitinib was approved by the FDA inMay of 2003. Gefitinib is used alone (monotherapy) for the treatment ofpatients with a certain type of lung cancer, i.e non-small cell lungcancer (NSCLC).

Approvals were based on two phase II trials, IDEAL 1 and 2, which showedIRESSA to be an effective treatment for many patients withpreviously-treated advanced NSCLC. Approximately 50% of patients in theIDEAL trials achieved tumor shrinkage or stabilization of their tumorand the drug was found to be generally well tolerated with the mostcommonly reported adverse drug reactions being mild-to-moderate skinrash and diarrhea. However, Interstitial Lung Disease (ILD), which maybe acute in onset, has been observed in patients receiving IRESSA, andsome cases have been fatal. Patients with concurrent idiopathicpulmonary fibrosis/interstitial pneumonia/pneumoconiosis/radiationpneumonia/drug-induced pneumonia have been observed to have an increasedrate of mortality from this condition.

In December 2004, AstraZeneca announced the results of the phase IIIISEL (IRESSA® Survival Evaluation in Lung Cancer) study which comparedIRESSA® with placebo in patients with advanced NSCLC who had failed oneor two prior chemotherapy regimens. ISEL showed some improvement insurvival with IRESSA® but this failed to reach statistical significance,compared with placebo, in the overall population or in patients withadenocarcinoma. Pre-planned subgroup analyses showed a statisticallysignificant increase in survival with IRESSA®, compared with placebo, inpatients of Asian origin and in patients who had never smoked. Inaddition, exploratory analyses of biomarker data from ISEL havesuggested that high EGFR gene copy number is a strong predictor ofbenefit with IRESSA® in pre-treated advanced NSCLC.

Following the announcement of the ISEL data, AstraZeneca voluntarilywithdrew the European submission for IRESSA® and regulatory authoritiesin the USA and Canada limited the use of IRESSA® to those patientsalready experiencing benefit from the drug. In the Asia Pacific region,due to the molecular differences in lung cancer, IRESSA® has become anestablished therapy for pre-treated advanced NSCLC and use of the drugin the first-line advanced setting is now being studied in a large phaseIII pan-Asian trial known as the IPASS study.

From the ISEL results, and broad clinical experience, it is clear thatIRESSA® is an effective treatment for some advanced NSCLC patients.AstraZeneca is now focused on identifying those NSCLC patients who aremost likely to benefit from the drug.

Since IRESSA® targets signaling pathways that appear to play a majorrole in the growth of many solid tumors it therefore may have atherapeutic potential in a broad range of cancers. Ongoing investigationof this potential includes clinical trials in head and neck cancer andbreast cancer.

Materials and Methods

1,2-Dioeoyl-3-trimethylammonium propane (DOTAP), dioleolylphosphatidylethanolamine (DOPE), and N-maleimido-phenylbutyrate DOPE (MPB-DOPE) werepurchased from Avanti Polar Lipids (Alabaster, Ala.).

Cell Lines and Culture

The human prostate cancer cell line DU145 (HTB-81) was obtained from theAmerican Type Culture Collection (ATCC; Manassas, Va.). DU14S wascultured in Eagle minimum essential medium with Earls salts (EMEM)supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mML-glutamine, and 50 μg/mL each of penicillin, streptomycin, andneomycin. The human melanoma cell line MDA-MB-435 and human breastcancer cell line MDA-MB-231 cell line were cultured in improved MEM(IMEM) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and50 μg/mL each of penicillin, streptomycin, and neomycin. EMEM waspurchased from MediaTech (Herndon, Va.) and the other cell culture mediaand ingredients were obtained from Biofluids (Rockville, Md.).

Preparation of TfRscFv/LipA/Geftinib (IRESSA®) Complexes

Cationic liposomal formulation LipA (DOTAP:DOPE or DDAB:DOPE at a 1:1 to1:2 molar ratio) were prepared using the ethanol injection method asdescribed throughout. TfRscFv/LipA/Geftinib (IRESSA®) complexes wereprepared as follows. After 10 minutes incubation with rotation orstirring at room temperature of a mixture of LipA and TfRscFv (ratio ofTfRscFv to LipA, 1:1 to 1:40 (wt/wt), more suitably 1:10 to 1:30 wt/wt),Geftinib (IRESSA®) at the appropriate concentration, was added mixed byinversion or stirring at room temperature and incubated for 10 minutes.For animal injection, dextrose or sucrose was added to each sample to afinal concentration of 1% to 20%, more suitably 5-10%. The molar ratioof Geftinib (IRESSA®) to Liposome was from 0.2:7 to 14:7, more suitably3.5:7 to 7:7, most suitably 7:7. The sizes of the complexes weredetermined by dynamic light scattering at 25° C. with a ZETASIZER®3000HS system (Malvern, United Kingdom).

In Vitro Cell Viability with the TfRscFv/LipA/Geftinib (IRESSA®) Complex

For in vitro cytotoxicity studies, 2.5 to 3.5×10³ cells/well in 100 μLof the appropriate growth medium of each cell line were plated in a96-well plate. After 24 hours, the cells were washed with serum-freemedium, overlaid with 100 μL of TfRscFv/LipA/Geftinib (IRESSA®) complexor free Geftinib (IRESSA®) in serum-free medium in increasingconcentrations, incubated for 4-6 hours, suitably 5 hours, and thensupplemented with FBS. The cells were then incubated for an additional24-72 hours, suitably 72 hours at 37° C. in a humidified atmospherecontaining 5% CO₂. Afterward, the wells were washed with IMEM withoutphenol red and the cell-viability XTT-based assay was performedaccording to the manufacturer's protocol (Boehringer Mannheim,Indianapolis, Ind.). In the presence of an electron-coupling reagent,XTT, sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonate is converted into orange formazan by dehydrogenase in themitochondria of living cells. The formazan absorbance, which correlatesto the number of living cells, was measured at 450 nm using a microplatereader (Molecular Devices, Menlo Park, Calif.). The IC₅₀ yielding 50%growth inhibition was interpolated from the graph of the log of drugconcentration versus the fraction of surviving cells.

In Vitro Chemosensitization

For the chemosensitization study, 2.5˜3.5×10³ cells/well in 100 μL wereseeded in a 96-well plate. After 24 hours, the cells were washed withserum-free medium, overlaid with 100 μL of TfRscFv/LipA/Geftinib(IRESSA®); complex or free Geftinib (IRESSA®) at 8 to 15 μM Geftinib(IRESSA®), incubated for 4-6 hours, suitably 5 hours, and then FBS wasadded to each well. The cells were incubated for an additional 24-72hours, suitably 19 hours, followed by the addition of the appropriatesupplemented medium with or without chemotherapeutics in increasingconcentrations, and incubation continued for approximately 24-72 hours,suitably 48 hours. The chemotherapeutic drugs used were docetaxel(TAXOTERE®; Aventis Pharmaceuticals, Bridgewater, N.J.), andmitoxantrone (NOVANTRONE® Immunex Corp., Seattle Wash.). The XTT assayswere performed to assess the degree of sensitization to thechemotherapeutics, and IC.sub.50 values of each cell were calculated.Fold sensitization equals the following: IC₅₀ untransfected/IC₅₀ eachcomplex.

Results

In Vitro Optimization of the Molar Ratio of TfRscFv/LipA/Geftinib(IRESSA®) Complex

The TtRscFv/LipA/Geftinib (IRESSA®) complex was prepared and the molarratio of the small molecule to liposome of the TfRscFv/LipA/Geftinib(IRESSA®) complex was optimized. The cell killing effect of complexedand free Geftinib (IRESSA®) at different ratios of Geftinib (IRESSA®) toLipA on MDA-MB-231 human breast cancer cells was compared. The ratio ofTfRscFv to LipA was 1:30 (wt/wt). 3×10³ cells/well were seeded in a96-well plate and treated after 24 hours with TfRscFv/LipA/Geftinib(IRESSA®) complexes, or free Geftinib (IRESSA®). The XTT assays wereperformed 72 hours after treatment to assess cytotoxicity, and the IC0values (the drug concentration yielding 50% growth inhibition) werecalculated from the concentration-cell viability curve. FIG. 44 showsthe IC₅₀ values for each ratio. At the range of the molar ratio ofGeftinib (IRESSA®) to liposome of 3:5˜7:7, a decrease in the ICs valueswere observed in cells treated with the complexed Geftinib (IRESSA®) incomparison with cells treated with free Geftinib (IRESSA®).

Comparison of Encapsulated and Free Geftinib (IRESSA®)

The effectiveness of TfRscFv/LipA/Geftinib (IRESSA®) compared to that offree Geftinib (IRESSA®) on cell survival was assessed via XTT assay (asdescribed herein) in a human breast cancer cell line (MDA-MB-231) (FIG.45A), a human melanoma cell line (MDA-MB-435) (FIG. 45B), and a humanprostate cancer cell line (DU145) (FIG. 45C). The molar ratio ofGeftinib (IRESSA®) to LipA in each complex was 7 to 7 (equivalent to1:1). The ratio of single-chain antibody fragment to liposome in eachcomplex was 1:30 (w:w). As demonstrated in FIG. 45A, treatment ofMDA-MB-231 cells with liposome complex delivered Geftinib (IRESSA®)resulted in a 2 fold increase in cell killing as compared to freeGeftinib (IRESSA®).

Moreover, in MDA-MB-435 cells a 1.5 fold increase in response wasobserved when the Geftinib (IRESSA®) was delivered as part of thecomplex of this invention (scL-IRESSA®), as compared to free Geftinib(IRESSA®) (FIG. 45B).

Similarly, in human prostate cancer cells, (FIG. 45C) Geftinib (IRESSA®)delivered via the liposome complex had a significantly greater effect oncell kill than when delivered in ‘free’ form. There was a 1.7 foldimprovement with TfRscFv/LipA/Geftinib (IRESSA®) over free smallmolecule.

Chemosensitization by TfRscFv/LipA/Geftinib (IRESSA®)

The ability of Geftinib (IRESSA®), delivered either ‘free’ or vialiposome complex (scL-IRESSA®), to sensitize tumor cells to first linechemotherapeutic agents was also assessed by XTT assay (FIG. 46A-C).Three different human cancer cell lines were treated with 8-15 uM freeor complexed Geftinib (IRESSA®) followed by addition of increasing dosesof chemotherapeutic agent. The molar ratio of Geftinib (IRESSA®) to LipAin each complex was 7 to 7 (equivalent to 1:1). The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). As shown in FIG. 46A, at a 12 uM Geftinib (IRESSA®) concentrationthere is greater than 2 fold increase in response to thechemotherapeutic agent (docetaxel) TAXOTERE® when delivered by theGeftinib (IRESSA®) liposome complex (scL-IRESSA®), as compared to whenit is administered as free Geftinib (IRESSA®).

Similarly in human melanoma cells (MDA-MB-435), Geftinib (IRESSA®)delivered by the liposome complex (scL-IRESSA®) at 15 .mu.M enhanced theresponse of the cells to (docetaxel) TAXOTERE® by 2.2 fold (FIG. 46B)compared to free Geftinib (IRESSA®) which did not sensitize the cells atall to the chemotherapeutic agent. The molar ratio of Geftinib (IRESSA®)to LipA in each complex was 7 to 7. The ratio of single-chain antibodyfragment to liposome in each complex was 1:30 (w:w).

The effect of liposome complex delivery of Geftinib (IRESSA®) was alsoassessed in human prostate cancer cell line DU145. The molar ratio ofGeftinib (IRESSA®) to LipA in each complex was 7 to 7. The ratio ofsingle-chain antibody fragment to liposome in each complex was 1:30(w:w). As was observed in the other tumor cells, there was a significantincrease in the level of sensitivity to chemotherapeutic agentMitoxantrone when Geftinib (IRESSA®) was delivered via the liposomecomplex of the present invention (scL-IRESSA®) (FIG. 46C). At 8 μM,there was an almost 5 fold increase in the response of DU145 toMitoxantrone with liposome complexed Geftinib (IRESSA®) over that seenwith free Geftinib (IRESSA®).

Thus, here again as with the other small molecules in the previousexamples, we have shown in multiple tumor cell types and with variouschemotherapeutic agents that the delivery and efficacy of the smallmolecule Geftinib (IRESSA®) is enhanced when it is encapsulated in thecomplex of this invention. Since the three tumor types shown in thisexample to be more sensitized to Geftinib (IRESSA®) when complexed asdescribed in this invention, as compared to free Geftinib (IRESSA®) arenot tumor types in which the use of this small molecule has been foundto be effective, it is an unexpected finding that these tumor cellsrespond so well to this invention.

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All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. An antibody- or antibody fragment-targetedcationic immunoliposome complex comprising a cationic liposome, anantibody or antibody fragment, and a small molecule inhibitor directedagainst a non-tumor disease, wherein said antibody or antibody fragmentis complexed with said cationic liposome, but is not chemicallyconjugated to said cationic liposome, wherein said antibody or antibodyfragment does not comprise a lipid tag, and wherein said antibody- orantibody fragment-targeted cationic immunoliposome is about 50-500 nm insize.
 2. The cationic immunoliposome complex of claim 1, wherein saidsmall molecule inhibitor is encapsulated within said cationic liposome.3. The cationic immunoliposome complex of claim 1, wherein said smallmolecule inhibitor is contained within a hydrocarbon chain region ofsaid cationic liposome.
 4. The cationic immunoliposome complex of claim1, wherein said small molecule inhibitor is associated with an inner orouter monolayer of said cationic liposome.
 5. The cationicimmunoliposome complex of claim 1, wherein said antibody fragment is asingle chain Fv fragment.
 6. The cationic immunoliposome complex ofclaim 1, wherein said antibody fragment is an anti-transferrin receptorsingle chain Fv (TfRscFv).
 7. The cationic immunoliposome complex ofclaim 1, wherein said cationic liposome comprises a mixture of one ormore cationic lipids and one or more neutral or helper lipids.
 8. Thecationic immunoliposome complex of claim 1, wherein said antibody orantibody fragment and said cationic liposome are present at a ratio inthe range of about 1:20 to about 1:40 (w:w).
 9. The cationicimmunoliposome complex of claim 1, wherein said cationic liposomecomprises a mixture of dioleoyltrimethylammonium phosphate withdioleoylphosphatidylethanolamine and cholesterol, a mixture ofdioleoyltrimethylammonium phosphate with cholesterol, a mixture ofdimethyldioctadecylammonium bromide withdioleoylphosphatidylethanolamine and cholesterol, a mixture ofdimethyldioctadecylammonium bromide withdioleoylphosphatidylethanolamine, a mixture ofdimethyldioctadecylammonium bromide with cholesterol, or a mixture ofdioleoyltrimethylammonium phosphate withdioleoylphosphatidylethanolamine.
 10. The cationic immunoliposomecomplex of claim 1, wherein said small molecule inhibitor and saidcationic immunoliposome are present at a molar ratio in the range ofabout 0.2:7 to about 14:7 (small molecule inhibitor:immunoliposome). 11.The cationic immunoliposome complex of claim 1, wherein small moleculeinhibitor and said cationic immunoliposome are present at a molar ratioof about 7:7 (small molecule inhibitor:immunoliposome).
 12. The cationicimmunoliposome complex of claim 1, wherein said small molecule inhibitoris directed against a neurodegenerative disorder.